JP5715641B2 - Method for diagnosing and monitoring cardiac ischemia in a patient with acute chest pain and no myocardial infarction - Google Patents

Description

  The present invention relates to a method for diagnosing an ischemic condition in a subject who shows signs and symptoms of general acute chest pain or acute coronary syndrome (non-ST elevation) but does not meet the diagnostic criteria for myocardial infarction and It relates to a method for monitoring the course of the severity of ischemic conditions. The invention further relates to a method for identifying a subject that is likely to receive a cardiac intervention and a method for determining a cardiac intervention. Also included are kits or devices for performing the methods of the invention. The method of the present invention measures the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) and optionally hepatocyte growth factor (HGF) in a sample of the subject, and optionally sFLT-1 and optionally HGF. Based on comparing the amount of at least one reference amount. Kits or devices for performing the methods of the present invention are also included in the present invention.

  The purpose of modern medicine is to provide individualized or individualized treatment plans. They are treatment plans that take into account the individual requirements or risks of the patient. A particularly important risk is the presence of cardiovascular complications, especially acute cardiovascular events. Cardiovascular complications belong to the main cause of morbidity and mortality in the Western Hemisphere. For individual treatment of a person suffering from cardiovascular complications, a reliable diagnosis has a significant impact on the success of the person's treatment. This is particularly important for patients presenting with signs and symptoms of acute coronary syndrome (ACS).

  The clinical symptoms of acute coronary syndrome are thought to be caused by acute myocardial infarction. Patients with chest pain or with signs and symptoms of unstable angina or acute coronary syndrome (ACS) often visit their doctor as an emergency or arrive in the emergency room. The clinical evaluation of these patients includes a history of evidence of existing cardiovascular disease or their risk factors, analysis of the types of symptoms described above, and clinical signs associated with acute coronary syndromes such as pulmonary edema, hypotension and / or Or evidence of tachycardia or bradycardia (Circulation 106: 1893, 2002 / Task force on Practice guidelines of the American Heart Association). In addition, ECG and laboratory tests are performed.

  Acute chest pain is the main symptom of ACS. However, chest pain is not specific for cardiovascular disease or ACS. Symptoms of chest pain can result from vascular disorders such as pulmonary embolism, aortic detachment or pulmonary hypertension, or lung diseases such as pleurisy, pneumonia, tracheobronchitis and spontaneous pneumothorax. Symptoms of acute chest pain can also be from gastrointestinal disorders such as esophageal reflux, peptic ulcer, gallbladder disease and pancreatitis, and musculoskeletal causes of acute pain include chondritis, cervical disc disease, trauma or contusion It is. Shingles can also cause acute chest pain. Furthermore, panic disorder needs to be considered as a differential diagnosis.

  Clinically, cardiac acute chest pain can be identified by clinical features such as retrosternal chest compressions or burning or malaise that radiates irregularly to the neck, jaw, upper abdomen, shoulder or left arm. The severity of acute chest pain often increases from angina to unstable angina and myocardial infarction. Facilitating causes include physical and emotional stress or the common cold. Severity is also associated with chest pain duration ranging from less than 2 minutes in angina to more than 30 minutes in myocardial infarction. Although these symptoms are quite characteristic in severe cases, it may be difficult to distinguish mild cardiac chest pain cases from non-cardiac causes (Braunwald Heart Disease, Chapter 49, page 1196).

  Patients with signs of acute coronary syndrome are at significantly increased risk of irreversible heart injury or even death, and therefore need to be identified in patients with atraumatic chest symptoms (Morrow et al., National academy of clinical biochemistry guidelines: Clinical characteristics and utilization of biochemical markers in acute coronary syndrome, 2007, Circulation; 115; 356-375). Acute coronary syndrome is caused by sudden coronary artery occlusion, and blood supply to the connected myocardium, heart muscle region is markedly suppressed or blocked, leading to ischemia (lack of blood supply).

  In the case of significant and / or persistent ischemia, the heart tissue becomes necrotic. Myocardial infarction (MI) (also called heart attack) is also known as cell necrosis of the myocardium (heart tissue) due to ischemia, as described by: The Joint ESC / ACCF / AHA / WHF Task Force for the Redefinition of Myocardial Infarction (The Joint European Society of Cardiology / American College of Cardiology Committee: Universal definition of myocardial infarction, European Heart Journal (2007), 28, 2525-2538).

  Thrombus is the most common cause of occlusion of coronary arteries that have generally been partially narrowed by atheroma. Atheromas can rupture or rupture, releasing substances that make platelets more viscous and can promote thrombus formation. In many cases, the thrombus dissolves naturally in about one day. However, some heart injury may have already occurred by this point.

  Assessment of medical history (history of coronary artery disease CAD) is one criterion for the diagnosis of patients presenting with ACS symptoms (eg chest pain over 20 minutes). These patients are further diagnosed with an electrocardiogram (ECG) and troponin test. If the initial ECG is undiagnosable at the time of arrival and the troponin test results do not meet the diagnostic criteria for non-ST elevation myocardial infarction, the procedure is repeated after 4-8 hours. If the ECG and troponin measurements still do not meet the diagnostic criteria for myocardial infarction, the patient is discharged with a diagnosis of excluding myocardial infarction.

  An electrocardiogram (ECG) provides important information for diagnosis. In particular, when ECG shows an increase in the ST portion, ST-elevated myocardial infarction (STEMI) is diagnosed. If ECG does not show an elevation of the ST moiety, a non-ST elevation MI (NSTEMl) is diagnosed if cardiac troponin is detected in each patient sample. Patients whose ECG is not diagnosable and whose cardiac troponin levels are less than an amount indicative of myocardial infarction are suspected of having unstable angina (UAP). Unstable angina and NSTEMl are closely related and are thought to share similar clinical symptoms. However, they differ in their severity. NSTEMl is distinguished from unstable angina by ischemia causing irreversible myocardial damage that can be detected by biomarkers of myocardial necrosis (Morrow et al., Cited above). In all the above cases, ie STEMI, NSTEMI and UAP, the patient is treated according to his diagnosis.

  If ECG (electrocardiogram) shows ST elevation, it is diagnosed as ST elevation myocardial infarction and the patient is considered for evaluation of reperfusion therapy. If ECG containing, including ECG ST or T wave changes, is still undiagnosable, the final diagnosis is revealed by the result of troponin T or I, which can diagnose non-ST elevation myocardial infarction. Most patients with signs or symptoms of chest pain or acute coronary syndrome exhibit diagnosable ECG and troponin results that do not meet the Non STEMI criteria according to current recommendations (The Joint European Society of Cardiology / American College of Cardiology Committee: Universal definition of myocardial infarction, European Heart Journal (2007), 28, 2525-2538). Such patients are associated with myocardial infarction, but often within the first 4-6 hours after the onset of signs / symptoms, i.e., the amount of circulating blood in the serum / plasma that begins to release cardiac specific necrosis markers from the myocardium It may be in the state before Other groups of patients may have chest pain that is completely unrelated to heart failure and therefore will not develop cardiac necrosis as indicated by characteristic ECG changes or increased troponin levels. Such patients will be finally diagnosed with non-cardiac chest pain. In a subgroup of patients, myoglobin (myoglobulin) and / or cardiac fatty acid binding protein (H-FABP) testing will show evidence of myocardial infarction, possibly diagnosed later by increased troponin associated with ECG changes .

  As noted above, current efforts in patients with chest pain or presenting symptoms are aimed at identifying or excluding myocardial infarction. So far, ECG and troponin have been key points for positive diagnosis of myocardial infarction or its exclusion.

  Troponin is a structural protein of the myocardium and is released upon necrosis or apoptosis of myocardial tissue. Furthermore, troponin is believed to be specific for myocardial tissue, and thus elevated levels of troponin in the circulation are considered indicators of myocardial infarction. Unfortunately, troponin levels only rise 4 to 6 hours after myocardial infarction, resulting in delayed diagnosis. This delay in recognizing myocardial infarction can result, for example, in delaying treatment with PCI, and therefore myocardium that may have been rescued by earlier intervention may be necrotic.

  Attempts to overcome this delayed recognition of myocardial infarction included the use of myoglobin or cardiac fatty acid binding proteins. Heart fatty acid binding protein (H-FABP) is a low molecular weight cytoplasmic protein and is present in large amounts in the myocardium. It has been observed that H-FABP is already released from the myocardium when the myocardium loses its function, i.e. long before the myocardium becomes necrotic. It has been clearly demonstrated that H-FABP levels above 5700 pg / ml are indicators of future troponin elevation and myocardial infarction, and H-FABP levels below 2500 pg / ml have been found to be independent of MI. Reference: WO 2008/145689.

  Similarly, myoglobin is another molecule that enters the circulation early after myocardial infarction. A myoglobin concentration above 77 ng / ml has been shown to be an indicator of future troponin elevation and thus myocardial infarction. On the other hand, if the myoglobin concentration is less than 55 ng / ml, there will be no future troponin expression, and therefore no possibility of infarction; see WO 2009/033831.

  Therefore, the measurement of myoglobin and / or H-FABP in serum or plasma is useful to compensate for some of the limits of troponin measurement, ie, that troponin appears late in the circulation (after 4-6 hours). Yes, if the appearance of troponin is predicted by myoglobin or cardiac fatty acid binding protein, it will aid in the early diagnosis of MI and thus allow intervention.

  Patients with symptoms of chest pain or acute coronary syndrome are usually discharged when symptoms resolve and they do not meet MI criteria. However, such patients may still have a high risk of myocardial infarction, as recently shown by serial ECG recordings (Tvivoni et al J. Am Coll Cardiol 53, 2009, 1422-24, Scirica Am Coll Cardiol 53, 2009, 1411-1421). In this study, ST drops as short as 1 minute and as small as ½ mm showed temporary ischemia and poor prognosis. However, this method is not easily applicable, requires computer-aided technology, and can only be implemented as a prospect. This highlights the severity of recognizing events prior to ischemia, necrosis and metabolic myocardial abnormalities.

WO 2008/145689 WO 2009/033831

Circulation 106: 1893, 2002 / Task force on Practice guidelines of the American Heart Association Braunwald Heart Disease, Chapter 49, page 1196 Morrow et al., National academy of clinical biochemistry guidelines: Clinical characteristics and utilization of biochemical markers in acute coronary syndrome, 2007, Circulation; 115; 356-375 The Joint ESC / ACCF / AHA / WHF Task Force for the Redefinition of Myocardial Infarction (The Joint European Society of Cardiology / American College of Cardiology Committee: Universal definition of myocardial infarction, European Heart Journal (2007), 28, 2525-2538) Tvivoni et al J. Am Coll Cardiol 53, 2009, 1422-24 Scirica Am Coll Cardiol 53, 2009, 1411-1421

  In summary, there is an urgent need for a method of identifying ischemia in patients with signs or symptoms of chest pain or acute coronary syndrome, and determining the extent of ischemia, its duration, and functional abnormalities associated with ischemia. Has been. These methods should also be able to rule out ischemia and further classify patients with signs or symptoms of chest pain or acute coronary syndrome, thereby providing improved diagnostic and therapeutic work-up.

  Therefore, diagnostic and prognostic means and method capable of reliable and rapid ischemia diagnosis in a subject showing signs and symptoms of acute coronary syndrome and having a cardiac troponin amount lower than a level indicative of myocardial infarction There is an obvious demand for Preferably, the individual will also have a non-diagnosable ECG, preferably an ECG that has not shown ST elevation. These measures and methods should not only be able to diagnose ischemia, but should also be able to assess the extent and changes in ischemia and assist in distinguishing between cardiac and non-cardiac causes of chest pain Should. In addition, this method is used in patients with cardiac chest pain and at other levels of cardiac biomarkers, such as troponin (but also myoglobin or H-FABP in some cases) below the diagnostic baseline characteristic of ischemia. ) Should help identify subgroups of patients exhibiting). Furthermore, ischemia can be quantified in cases of sustained or continuous ischemia and events of recurrent ischemic episodes. These methods and measures should also be able to identify subjects who are likely to receive cardiac intervention and determine if the subject's cardiac intervention is appropriate and, if affirmative, which therapy should be selected It is. These methods and means should avoid at least some of the shortcomings of the current technology shown above.

  Accordingly, the technical problem underlying the present invention should be viewed as providing means and methods that meet the above needs.

  The technical problem is solved by the claims and the embodiments described below.

  The present invention is a method for diagnosing an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction, comprising soluble fms-like in the subject's sample Methods are provided that are based on comparing the amount of tyrosine kinase-1 (sFLT-1) or a variant thereof to at least one reference amount. The method can include at least one of the following steps: a) measuring the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof in the subject sample, and b) step a SFLT-1 or a variant thereof measured in step 1) is compared with at least one reference amount, and c) based on the information obtained in step b), preferably based on the information obtained in steps a) and b) To diagnose an ischemic condition.

Accordingly, the present invention is a method for diagnosing an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) measuring the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof in the subject sample;
b) comparing the amount of sFLT-1 or its variant measured in step a) with at least one reference amount, and c) based on the information obtained in step b), preferably obtained in steps a) and b) And a method comprising diagnosing an ischemic condition based on the obtained information.

The present invention further provides a method for diagnosing an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) comparing the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof measured in the subject sample with at least one reference amount, and b) based on the information obtained in step a) It relates to a method comprising diagnosing an ischemic condition.

The present invention further provides a method for diagnosing an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) a method comprising diagnosing an ischemic condition based on a comparison of the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof measured in the subject sample with at least one reference amount About.

  The present invention is a method for monitoring an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction, wherein the subject has at least two different time points. Provided is a method based on comparing the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof measured in a sample to at least one reference amount. The method can include at least one of the following steps: a) Measuring the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof in the subject sample at at least two different time points B) comparing the amount of sFLT-1 or its variant measured in step a) with at least one reference amount, and c) based on the information obtained in step b), preferably steps a) and b) Based on the information obtained in step 1, the ischemic state at at least two different time points is diagnosed.

Accordingly, the present invention is also a method for monitoring an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) measuring the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof in the subject sample at at least two different time points, and b) sFLT-1 or variant thereof measured in step a) And c) based on the information obtained in step b), preferably based on the information obtained in steps a) and b) at at least two different time points. A method comprising diagnosing an ischemic condition of the patient and thereby monitoring the ischemic condition.

The present invention further provides a method for monitoring an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) comparing the amount of sFLT-1 or variant thereof in the subject sample measured at at least two different time points with at least one reference amount, and b) ischemia based on the information obtained in step a) It relates to a method comprising diagnosing a condition and thereby monitoring an ischemic condition.

The present invention further provides a method for monitoring an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) Ischemic status based on a comparison of the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof in the subject sample measured at at least two different time points with at least one reference amount Relates to a method comprising monitoring.

  In an embodiment of the method of the invention as described above, in an additional step aa) the amount of hepatocyte growth factor (HGF) or variant thereof in the subject sample is measured, and in step bb) at least one reference amount for HGF; Compare. Thus, in step c), a measured amount of sFLT-1 or a variant thereof and HGF or a variant thereof, and a comparison between the amount of sFLT-1 and at least one reference amount for sFLT-1, and an amount of HGF and at least for HGF An ischemic condition is diagnosed based on a comparison with one reference amount. Preferably, the amount of sFLT-1 and then the amount of HGF are measured first, but it is contemplated that the amounts of sFLT-1 and HGF are measured in any order; ie, simultaneously or first sFLT-1 and then HGF Or first HGF and then sFLT-1.

  In yet another aspect of the invention, in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction, the level of hepatocyte growth factor HGF or a variant thereof in the subject's sample is determined. The ischemic state is diagnosed by measuring. Similarly, in yet another aspect of the present invention, hepatocyte proliferation in a sample of the subject at least two different time points in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction The ischemic state is monitored by measuring the level of factor HGF or variants thereof. HGF is an independent ischemic marker that conveys information about ischemia, and in many cases it has been shown to be as good as the information obtained with sFLT-1. Thus, in some cases sFLT-1 provides better information; in some cases HGF provides better information; and in some cases it can be said that the information in both markers is equivalent.

  In an embodiment of the method of the invention as described above, in an additional step aa) the amount of fms-like tyrosine kinase-1 (sFLT-1) or variant thereof in the subject sample is measured, and in step bb) the fms-like tyrosine kinase -1 (sFLT-1) compared to at least one reference quantity. Thus, in step c), the measured amount of sFLT-1 or a variant thereof and HGF or a variant thereof, and the comparison of the amount of sFLT-1 with at least one reference amount for sFLT-1 and the amount of HGF and at least one for HGF An ischemic condition is diagnosed based on a comparison with two reference quantities. Preferably, the amount of HGF is first measured, then the amount of sFLT-1, but it is contemplated that the amounts of sFLT-1 and HGF are measured in any order; ie, simultaneously or first sFLT-1 and then HGF Or first HGF and then sFLT-1.

Accordingly, the present invention is a method for diagnosing an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) measuring the amount of hepatocyte growth factor HGF or variant thereof in the subject sample;
b) comparing the amount of HGF or variant thereof measured in step a) with at least one reference amount, and c) based on the information obtained in step b), preferably information obtained in steps a) and b) To a method comprising diagnosing an ischemic condition.

The present invention further provides a method for diagnosing an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) comparing the amount of HGF or variant thereof measured in the subject sample with at least one reference amount, and b) diagnosing an ischemic condition based on the information obtained in step a) .

The present invention further provides a method for diagnosing an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) to a method comprising diagnosing an ischemic condition based on a comparison of the amount of HGF or variant thereof measured in the sample of the subject with at least one reference amount.

The present invention also provides a method for monitoring an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) measuring the amount of hepatocyte growth factor HGF or variant thereof in the subject sample at at least two different time points; and b) determining the amount of HGF or variant thereof measured in step a) as at least one reference amount. And c) diagnosing ischemic conditions at at least two different time points based on the information obtained in step b), preferably based on the information obtained in steps a) and b) To monitoring the ischemic condition.

The present invention further provides a method for monitoring an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) comparing the amount of HGF or variant thereof in the subject sample measured at at least two different time points with at least one reference amount, and b) determining ischemic status based on the information obtained in step a) It relates to a method comprising diagnosing and thereby monitoring ischemic conditions.

The present invention further provides a method for monitoring an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) to a method comprising monitoring an ischemic state based on a comparison of at least one reference amount with the amount of HGF or variant thereof in the subject's sample measured at at least two different time points.

  In a preferred embodiment, the amount of cardiac troponin is measured simultaneously with the measurement of sFLT-1 and / or HGF; the measurement of the amount of cardiac troponin precedes the measurement of sFLT-1 and / or HGF Also good. Thus, the ischemic state of an individual is preferably determined by measuring the amount of sFLT-1 and / or HGF, or optionally still other markers, simultaneously with or after the measurement of troponin. Thus, if the amount of cardiac troponin is measured prior to the measurement of sFLT-1 and / or HGF, the measurement of the amount of sFLT-1 and / or HGF is postponed until the amount of cardiac troponin is known, and myocardial infarction The amount of sFLT-1 and / or HGF is measured only when the amount of troponin is less than the amount generally recognized in the art as an indicator of MI, particularly NSTEMI. The amount of cardiac troponin can even be zero, i.e. not detectable with currently available tests.

  In yet another preferred embodiment, the ECG of each subject is measured simultaneously with the sFLT-1 measurement and / or the HGF measurement; the ECG measurement may be performed prior to the sFLT-1 measurement and / or the HGF measurement. . Accordingly, the ischemic state of the individual is determined by measuring the amount of sFLT-1 and optionally HGF, preferably simultaneously with or after the ECG measurement. Thus, if the ECG measurement is performed prior to the measurement of sFLT-1 and / or HGF, the measurement of the amount of sFLT-1 and / or HGF may be postponed until the ECG is recorded. Preferably, the amount of sFLT-1 and / or HGF is measured only when the subject's ECG does not show ST elevation. Preferably, if the ECG shows ST elevation, the subject will be considered as suffering from STEMI; this is generally performed according to the method of the invention—for the purpose of diagnosing ischemic conditions prior to MI— It means not.

  In relation to the above, the measurement of the amount of cardiac troponin and / or the measurement of ECG can be performed simultaneously or sequentially, including the measurement of sFLT-1 and / or HGF described above.

  Assessment of medical history (history of coronary artery disease CAD) is one diagnostic criterion for the diagnosis of patients presenting with ACS symptoms (eg, chest pain over 20 minutes). These patients are further diagnosed with the fulfillment of other diagnostic criteria, ie, on the one hand using an electrocardiogram (ECG) and on the other hand using a troponin test. If the initial ECG does not meet the diagnostic criteria for non-ST elevation myocardial infarction upon arrival, and if the troponin test results do not meet the diagnostic criteria for myocardial infarction, the procedure is repeated 4-8 hours later. If ECG and troponin measurements still do not meet the diagnostic criteria for myocardial infarction, the patient is discharged with a diagnosis of excluding myocardial infarction. The latest standard for diagnosis of myocardial infarction MI is The Joint ESC / ACCF / AHA / WHF Task Force for the Redefinition of Myocardial Infarction (The Joint European Society of Cardiology / American College of Cardiology Committee: Universal definition of myocardial infarction, above ).

  The method of the present invention is preferably an in vitro method. In addition, it can include other steps than those specified above. For example, yet another step may relate to sample pretreatment or evaluation of the results obtained by the method. The methods of the invention can also be used for diagnostic monitoring, confirmation and subclassification. The method can be carried out manually or assisted by automatic operation. Preferably, all or part of steps (a), (b) and / or (c) is performed automatically, eg a robotic device and sensing device suitable for measurement in step (a), or a computer in step (b) You may support by processing comparison.

  As used herein, the term “signs and symptoms of acute coronary syndrome” relates on the one hand to signs that can be indicative of ACS, but they can also occur in diseases other than ACS. Thus, this term is a sign that is not explicitly related to the occurrence of ACS, but is an indicator of the probability of ACS occurrence, thereby further confirming (rule in / rule out) Including those for which testing is required. An example of such a sign is chest pain. Of course, chest pain is a vascular disorder such as pulmonary embolism, aortic detachment or pulmonary hypertension, or lung disease such as pleurisy, pneumonia, tracheobronchitis and spontaneous pneumothorax; gastrointestinal diseases such as esophageal reflux, peptic ulcer, gallbladder disease and It can originate from pancreatitis, and musculoskeletal causes of acute pain include costal chondritis, cervical disc disease, trauma or contusion. Shingles can also cause acute chest pain. These forms of chest pain are often referred to as “non-cardiac chest pain”. On the other hand, this term relates to acute cardiac chest pain that develops symptoms such as retrosternal chest compression, or burning or malaise that irregularly radiates to the neck, jaw, upper abdomen, shoulder or left arm. The severity of acute chest pain often increases from angina to unstable angina and myocardial infarction. Facilitating causes include physical and emotional stress or the common cold. Severity is also related to the duration of chest pain ranging from less than 2 minutes for angina to more than 30 minutes for myocardial infarction. Although these symptoms are quite characteristic in severe cases, it may be difficult to distinguish mild cardiac chest pain cases from non-cardiac causes. Still other known symptoms of ACS include upper abdominal, arm, wrist or jaw discomfort or pain, unexplained nausea or vomiting, persistent shortness of breath, weakness, dizziness, dizziness, or fainting, and the like Any combination.

  However, the symptoms may not appear specific enough to make a reliable and proper diagnosis of ACS (or avoid misdiagnosis) in many cases. For example, chest pain may not be in a clear position. Other groups of patients may have chest pain that is completely unrelated to heart failure and therefore will not develop characteristic ECG changes, or cardiac necrosis that is indexed by increased troponin levels. Such a patient will be finally diagnosed with non-cardiac chest pain. The cardiologist may be able to include or exclude the occurrence of ACS based on the identification of symptoms typical of ACS and ignore symptoms that are not typical of ACS. However, this method is error prone and modern cardiology requires measurement of cardiac troponin and ECG in addition to interpretation of symptoms (and assessment of medical history) for each individual. Thus, the methods of the present invention include individuals with ACS signs and symptoms, particularly chest pain, where the signs / symptoms, particularly chest pain, may be related to ACS but also to disorders other than ACS. There is a possibility that there is no clear diagnosis of ACS; however, those symptoms, particularly chest pain, require further diagnosis / evaluation of the underlying disease to include or exclude ACS. In some cases, an individual applying the method of the present invention may exhibit distinct symptoms for ACS, such as intense chest pain that radiates to the arms or shoulders.

  As used herein, the term “diagnose” refers to an ischemic condition in a subject that exhibits signs and symptoms of coronary syndrome and has an amount of cardiac troponin that is less than the amount that is indicative of MI, particularly if the subject has a reversible heart. It means assessing, identifying, assessing or classifying whether or not you are suffering from an ischemic condition resulting in dysfunction or irreversible heart injury. The term “diagnose” refers to a physiologically healthy subject and possibly reversible heart function in a subject who exhibits signs and symptoms of coronary syndrome and preferably has a cardiac troponin amount less than that which is indicative of MI. It also represents identifying a subject suffering from ischemia that will result in failure or irreversible heart injury.

The diagnosis of ACS and the criteria applied for that diagnosis are generally known in the art and include, among other things, a medical history (history of coronary artery disease CAD) and an assessment of chest pain over about 20 minutes.
As used herein, the term “subjects who show signs and symptoms of acute coronary syndrome and do not meet the diagnostic criteria for myocardial infarction” may or may be associated with the onset or diagnosis of myocardial infarction. Included subjects with no signs and symptoms of acute coronary syndrome; however, those signs and symptoms shown are insufficient to diagnose myocardial infarction without leaving doubt about the diagnostic results. Diagnostic criteria for myocardial infarction is described by The Joint ESC / ACCF / AHA / WHF Task Force for the Redefinition of Myocardial Infarction (The Joint European Society of Cardiology / American College of Cardiology Committee: Universal definition of myocardial infarction, cited above) Has already been summarized earlier.

  Preferably, the amount of cardiac troponin is measured, and the subject exhibits an amount of cardiac troponin that is less than an amount indicative of myocardial infarction. Alternatively, an electrocardiogram (ECG) is recorded in the subject, and the ECG does not meet the criteria for myocardial infarction, preferably the patient does not meet the criteria for non-ST elevation MI. Even more preferably, the level of cardiac troponin is measured and an ECG is recorded, these two parameters do not meet the criteria for myocardial infarction; that is, the measured amount of troponin is less than the amount indicative of myocardial infarction, and / Or ECG. If the ECG does not meet the criteria for myocardial infarction at the time of arrival and if the troponin test results do not meet the diagnostic criteria for myocardial infarction at the time of arrival, this procedure is preferably performed after about 4 to about 8 hours. repeat. If the ECG and troponin measurements still do not meet the diagnostic criteria for myocardial infarction, the patient can be discharged with a diagnosis of excluding myocardial infarction.

  As used herein, the term “ischemic condition” relates to a condition of impaired blood supply that is not sufficient for metabolic demands on the affected tissue, particularly not sufficient for oxygen supply. Ischemia is associated with, can cause, or can cause changes in myocardial function, depending on the extent of ischemic myocardium and ischemic duration. After a single episode of ischemia lasting less than 2 minutes, myocardial function normalizes rapidly. As the duration and / or severity of ischemia increases, there is a temporary delay in functional recovery, which occurs despite the fact that blood flow has been restored. When a blood vessel is occluded for 15 minutes, a change in myocardial function of, for example, 6 hours occurs even when blood flow is restored. This reversible event is not associated with myocardial necrosis, preferably the term “ischemia” or “ischemic condition” results in reversible cardiac dysfunction and irreversible heart injury, as well as reversible cardiac dysfunction and irreversible heart injury. , Processes related to or responsible for them. Ischemia or ischemic conditions also includes heart injury associated with, causing or causing myocardial infarction.

  As used herein, the term “reversible cardiac dysfunction” is an impairment of the heart's pumping ability or activity and is completely reversible, preferably any significant, including a significant number of cardiomyocyte necrosis. It relates to what happens without leaving the structural deterioration in the heart. Preferably, the impairment of pumping ability or activity does not cause any significant injury to the subject's body. Reversible cardiac dysfunction can return immediately, for example within seconds or minutes, as in the case of very short ischemic periods. Other examples of reversible cardiac dysfunction include cardiac stunning. In these cases, the return may be delayed for hours or days.

If a significant percentage of the myocardium is ischemic, this can result in wall stretch and a rapid increase in natriuretic peptides.
In a further preferred embodiment of the invention, the amount of natriuretic peptides, ie BNP-type peptides belonging to group BNP and NT-proBNP, or ANP-type peptides belonging to group ANP and NT-proANP is measured. Preferably, ANP-type peptides, more preferably NT-proANP, are measured to diagnose the subject's possible circulatory disturbance.

  Therefore, although the cause is not related to ischemia, an increase in the amount of ANP-type sodium excretion increasing peptide compared to the reference amount is an indicator that the ischemic state is in a specific region of the myocardium, which is non-ischemic. Increased pumping demand on the myocardium and consequently circulatory disturbance.

  In the present invention, measurement of an ANP-type sodium excretion increasing peptide, preferably NT-proANP is more preferable than measurement of a BNP-type sodium excretion increasing peptide, such as NT-proBNP. This is because the ANP type peptide is released rapidly. After an increase in heart pressure and volume (wall extension), the amount of NT-proANP increases within 15-60 minutes, which allows rapid diagnosis of circulatory dysfunction. This is essential in the context of the present invention in order to be able to initiate a procedure that allows the rescue of myocardium suffering from ischemia but not yet necrotic.

  In contrast, BNP-type sodium excretion increasing peptides, especially NT-proBNP, finally increase 4-6 hours after the increase in heart pressure and volume (wall extension), which in many cases is for the purposes of the present invention. It is insufficient.

  In the present invention, the circulatory disorder is caused by the formation of an ischemic region in the myocardium that has maintained metabolism or a myocardial region that is dysfunctional (with a lower metabolism than a fully functional myocardium, It is assumed that it is related to or caused by the pumping at a lower performance that does not exceed the long-term pumping performance. As a result of the reduced pumping capacity or activity of the ischemic and / or dysfunctional myocardium, the remaining unimpaired myocardium must ensure body demand for blood supply and may have to work excessively. is there.

  In a preferred embodiment of the invention, subjects with dysfunctional myocardium and / or subjects suffering from STEMI can be excluded by measuring the amount of H-FABP and / or myoglobin. In general, this aspect is to determine whether the subject suffers from myocardial infarction, in particular NSTEMI, after cardiac troponin measurement and / or ECG measurement, and at steps a) and b) of the method of the invention. -1 and optionally measuring H-FABP and / or myoglobin prior to measuring the amount of HGF.

  In myocardium that has stopped functioning, myocardial function is suppressed to a quiescent state, but myocardial cells remain viable. LV dysfunction in a dysfunctional state can be reversible. Defunct myocardium is most common after the transition to reperfusion following ischemia (function is kept quiescent but perfusion is preserved). Ischemic episodes can be single or multiple, short or long-term, but never severe enough to cause injury.

"Irreversible heart injury" is generally known in the art and relates to heart injury associated with cell death, preferably cardiomyocyte necrosis, eg, due to a necrotic process.
An amount of H-FABP and / or myoglobin that is greater than a reference amount that includes the occurrence of MI in a sample of the subject defined in the present invention is an indicator of recent MI occurrence in that subject. In a subject defined in the present invention, an amount of myoglobin and optionally H-FABP that is less than a reference amount that excludes the occurrence of MI should be an indication that an MI infarction has not recently occurred; The subject may suffer from UAP. In the context of the present invention, subjects subject to the present invention whose myoglobin amount is between the above-mentioned reference amounts (a reference amount that includes recent MI occurrence and a reference amount that excludes recent MI occurrence) are subject to rediagnosis. It should be understood that it may be necessary. Preferably, it measures the amount of both myoglobin and H-FABP, where both amounts do not correspond, eg one amount is greater (or less) than each reference amount, whereas the other amount Can also be performed for subjects that are not more (or less) than each reference amount. In particular, in the subject defined in the present invention, an amount of myoglobin greater than about 77 ng / ml is an indicator of recent MI occurrence (inclusion), whereas an amount less than about 55 ng / ml is an indicator that MI has not recently occurred. (Excluded). Further, the sensitivity and specificity of the diagnosis based on the measurement of myoglobin in the sample of the subject defined in the present invention is at least 1 for H-FABP by measuring the amount of H-FABP in the sample of the subject in addition to the amount of myoglobin. It increases even more when compared to one reference amount. In particular, in the subject defined by the present invention, an amount of H-FABP greater than about 5700 pg / ml is an indicator of recent MI occurrence (included), whereas an amount less than about 2500 pg / ml has not recently produced MI. (Excluded).

  The term “diagnosing circulatory disorders” refers to an ANP-type peptide, preferably NT-proANP, in an amount greater than the amount considered to be indicative of a subject as defined in the present invention, i.e. not suffering from ACS and a healthy subject. It relates to evaluating whether or not a circulatory disorder has occurred in the subject. The term “circulatory disturbance” relates to an impairment in the ability or activity to pump the necessary amount of blood into the circulation to ensure that the heart (myocardium) or area thereof supplies the tissue with sufficient blood for metabolic demand. . In order to compensate for the disability, the myocardium must work harder, resulting in increased wall stress (and release of NT-proANP). For example, if an area of the myocardium is affected by ischemia, the pumping ability of the affected area is impaired. In order to ensure a sufficient blood supply, the myocardial pumping work in areas that are not particularly affected and maintain their full pumping capacity is enhanced. However, NT-proANP expression is stimulated because it must be overworked.

  To compensate for the lack of blood supply, the heart must work harder than normally required, resulting in increased stress on the heart wall (since the heart must be pumped more intensely). Such a disorder is not harmful as long as the underlying cause is removed before the irreversible heart injury occurs. There are various reasons for circulatory disturbance.

The term “subject” as used herein relates to an animal, preferably a mammal, more preferably a human.
As used herein, the term “simultaneously” refers to the measurement of a certain activity, preferably a marker used in connection with the present invention, at the same time. This is preferably because the measurement of one marker is slightly delayed from the measurement of the other marker, for example a few seconds or minutes, for example 1 minute, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes Includes measurements. It is essential that the measurement of the latter marker is not delayed so that its amount can change to different diagnostic results. “Measuring a marker” refers to taking a bodily fluid sample that measures the amount of marker therein, or measuring and detecting the amount of marker, or both.

  As used herein, the term “measuring (amount of marker) at least two different time points” shall include the measurement of the amount of marker at intervals, wherein the second and subsequent samples are: It will be taken at intervals to ensure effective monitoring of ischemic conditions. In general, the interval between each sample is about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 90 minutes, about 2 hours, about 3, 4, 5 or 6 hours. For example, the first sample is taken about 1 hour after symptoms of acute coronary syndrome or immediately after the subject is presented to the physician, and each subsequent sample is taken about 1 hour after the first sample. The number of samples taken will depend on the assessment of ischemia.

  The terms “acute coronary syndrome” (ACS) and ACS diagnostic criteria are understood and known to those skilled in the art. The term relates to a group of clinical symptoms caused by acute myocardial infarction. Ischemia itself results from the collapse of atherosclerotic plaques in the coronary arteries. It is known in the art that ACS may be accompanied by symptoms such as upper abdomen, arm, wrist or jaw discomfort or pain, especially chest pain, where chest pain lasts longer than 20 minutes. May spread radially to the arms, back or shoulders. Still other symptoms of an acute cardiovascular event may be unexplained nausea or vomiting, shortness of breath, weakness, dizziness, dizziness, sweating or fainting, and any combination thereof. In general, these clinical symptoms, particularly chest pain, occur suddenly; they may appear at rest or after a small effort. Furthermore, in the context of the present invention, the term “acute coronary syndrome” may relate to a suspected, probable or probable ACS; these terms indicate signs and symptoms consistent with ACS, but a diagnosis for it is reliably established. This is because it is often used for patients who do not (see: Morrow et al., Cited above). ACS patients may exhibit unstable angina (UAP) or these individuals may have myocardial infarction (MI). The MI may be ST-elevated MI (STEMI) or non-ST-elevated MI (NSTEMI). MI is classified as belonging to coronary heart disease CHD, and prior to this, other events that are also classified as belonging to CHD, such as unstable angina UAP, occur. The symptom of UAP is chest pain, which is alleviated by sublingual administration of nitroglycerin. UAP occurs due to partial occlusion of the coronary arteries, resulting in hypoxemia and myocardial infarction. In some cases, the obstruction is remarkably severe or complete, resulting in irreversible myocardial necrosis (a pathological condition that causes myocardial infarction). In general, STEMI is diagnosed by electrocardiogram measurement when the electrocardiogram (ECG) shows an increase in the ST section. UAP and NSTEMI can be distinguished by measuring cardiac troponin levels for at least 6 hours after the onset of ACS. If the amount of troponin is increasing (which is an index of myocardial damage), NSTEMI is estimated. MI may occur without obvious symptoms; that is, the subject does not exhibit any discomfort and does not have stable or unstable angina prior to MI. Left ventricular dysfunction (LVD) may occur following the onset of MI.

  The term “cardiac troponin” refers to all troponin isoforms expressed in cardiac cells, preferably subendocardial cells. These isoforms are well characterized in the art as described, for example, in Anderson 1995, Circulation Research, vol. 76, no. 4: 681-686 and Ferrieres 1998, Clinical Chemistry, 44: 487-493. It has been elucidated. Preferably, cardiac troponin represents troponin T and / or troponin I, most preferably troponin T. It should be understood that the methods of the present invention may measure troponin isoforms together, ie simultaneously or sequentially, or individually, ie without any other isoforms. is there. The amino acid sequences of human troponin T and human troponin I are shown in Anderson, cited above, and Ferrieres 1998, Clinical Chemistry, 44: 487-493.

  The term “cardiac troponin” also includes variants of the specific troponins mentioned above, ie preferably troponin I, more preferably troponin T. Such variants have at least the same essential biological and immunological properties as the particular cardiac troponin. In particular, they are of the same essential if they can be detected by the same specific assay described herein, eg, an ELISA assay using polyclonal or monoclonal antibodies that specifically recognize their cardiac troponin. Share biological and immunological properties. Furthermore, the variants described according to the invention should have different amino acid sequences due to substitutions, deletions and / or additions of at least one amino acid, wherein the variant amino acid sequence is still preferably the full length of a particular troponin. Across the amino acid sequence of that particular troponin and preferably at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about It should be understood that 95%, at least about 97%, at least about 98%, or at least about 99% are identical. The variant may be an allelic variant, or any other species-specific homolog, paralog or ortholog. In addition, variants described herein include specific cardiac troponins or fragments of variants of the above types; however, these fragments exhibit the essential immunological and biological properties described above. Have. Preferably, variants of cardiac troponin have immunological properties (ie, epitope composition) comparable to those of human troponin T or troponin I. Thus, the variant should be recognized by the means or ligands used to measure cardiac troponin levels. Such a fragment may be, for example, a degradation product of troponin. In addition, different variants are included due to post-translational modifications such as phosphorylation or myristylation. Preferably, the biological property of troponin I and variants thereof is the ability to inhibit actomyosin ATPase or to inhibit angiogenesis in vivo and in vitro, for example Moses et al. 1999 PNAS USA 96 (6): It can be detected based on the assay method described by 2645-2650). Preferably, the biological properties of troponin T and variants thereof are preferably that if a complex of troponin C, I and T or a complex formed by troponin C, troponin I and a troponin T variant is present The ability to form a complex with troponins C and I, the ability to bind calcium ions, or the ability to bind to tropomyosin.

  The term “cardiac troponin amount” as used herein relates to the concentration of cardiac troponin, preferably troponin T. Preferably, the term relates to the concentration of cardiac troponin in the subject's serum or plasma. “Cardiac troponin less than an amount indicative of myocardial infarction” is any amount of cardiac troponin starting from zero and including zero, which can be obtained by means and methods known in the art, eg, commercially available cardiac troponin It may relate to an amount detectable by the assay method. Preferably, “detectable amount of cardiac troponin” relates to a concentration above the minimum detection limit of the assay used to measure the amount of troponin. Preferably, the detectable amount of troponin relates to any concentration of 0.001 ng / ml, 0.002 ng / ml, 0.005 ng / ml, 0.0075 ng / ml, or 0.01 ng / ml or more. Good. More preferably, the detectable amount of cardiac troponin may relate to any concentration of 0.002 ng / ml or more. “Troponin amount serving as an index of myocardial infarction” relates to a troponin concentration serving as a generally accepted index of myocardial infarction.

  Troponin T was tested with a sensitive troponin T test using an ELECSYS 2010 analyzer (Roche Diagnostics, Mannheim, Germany). This inspection was performed according to the manufacturer's instructions. This test method has a measuring range of 3 to 10.000 ng / ml or pg / ml. The accuracy of this test method was found to be 0.8-2.6 percent depending on the troponin concentration in the sample.

Preferably, the amount of biomarker, preferably the amount of cardiac troponin, that is considered to be indicative of myocardial infarction, measured in the sample, exceeds the 95 ^th percentile concentration of the appropriate reference population, preferably 99 ^th percent Concentration above the point concentration (cut-off score). This amount is the recommendation made by The Joint ESC / ACCF / AHA / WHF Task Force for the Redefinition of Myocardial Infarction (The Joint European Society of Cardiology / American College of Cardiology Committee: Universal definition of myocardial infarction, cited above). Based. How to select the appropriate reference population, as well as a method of determining the 95 ^th and 99 ^th percentile concentration is known to those skilled in the art. It should be understood that this concentration may vary based on the assay used to measure cardiac troponin concentration and based on the reference population chosen. Preferred cardiac troponin levels indicative of MI for the present invention are at least about 0.05 ng / ml, at least about 0.075 ng / ml, at least about 0.099 ng / ml, at least about 0.1 ng / ml, at least about 0. The amount may be, but is not limited to, .2 ng / ml, and at least about 0.3 ng / ml.

In a preferred embodiment of the method of the present invention, the amount of troponin in a subject having a cardiac troponin amount less than the amount (as defined in the present invention) showing signs and symptoms of acute coronary syndrome and indicative of myocardial infarction, in particular troponin The amount of T is about 0.002 ng / ml or more and less than about 0.1 ng / ml. Above cited The Joint ESC / ACCF / AHA / WHF Task Force for the Redefinition of Myocardial Infarction 99 th percentile calculated in accordance with the requirements of a 0.14 ng / ml, which is the most preferred amount indicative of MI is there.

  The term “soluble (s) FLT-1” as used herein refers to a polypeptide that is a soluble form of the VEGF receptor FLT1. It was identified in the conditioned medium of human umbilical vein endothelial cells. The endogenous soluble FLT1 (sFLT1) receptor is chromatographically and immunologically similar to recombinant human sFLT-1 and binds [125I] VEGF with comparable high affinity. Human sFLT-1 has been shown to form a VEGF-stabilized complex with the extracellular domain of KDR / Flk-1 in vitro. Preferably sFLT1 represents human sFLT1. More preferably, human sFLT1 can be deduced from the amino acid sequence of Flt-1 set forth in Genebank accession number P17948, GI: 125361. The amino acid sequence of mouse FLT1 is shown in Genebank accession number BAA24499.1, GI: 2809071. Furthermore, variants described in accordance with the present invention should have different amino acid sequences due to substitutions, deletions and / or additions of at least one amino acid, where the amino acid sequence of the variant is still the amino acid sequence of a particular sFLT1. It should be understood that preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% are identical. The variant may be an allelic variant, a splice variant, or any other species-specific homolog, paralog or ortholog. Furthermore, variants described herein include specific sFLT1 or fragments of the above types of variants; however, these fragments have the essential immunological and biological properties described above. Preferably, the sFLT-1 variant has immunological properties (ie, epitope composition) and / or biological properties comparable to those of human sFLT-1. Thus, the variant should be recognized by the means or ligand used to measure the amount of sFLT-1. Such a fragment may be, for example, a degradation product of sFLT1. In addition, different variants are included due to post-translational modifications such as glycosylation, phosphorylation or myristylation. Preferably, the biological property of sFLT-1 is its ability to bind VEGF with high affinity and / or form a VEGF stabilized complex with the extracellular domain of KDR / Flk-1.

  In yet another aspect of the invention, for diagnosing myocardial infarction based on a comparison of the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof in a sample of a subject with at least one reference amount. The amount of sFLT-1 measured in the individual is used. The method can include at least one of the following steps: a) measuring the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof in the subject sample, and b) step a SFLT-1 or a variant thereof measured in step 1) is compared with at least one reference amount, and c) based on the information obtained in step b), preferably based on the information obtained in steps a) and b) Thus, myocardial infarction, particularly non-ST elevation myocardial infarction is diagnosed. This is particularly advantageous when the individual can be observed for a long time, preferably longer than 6 hours, more preferably longer than 8 hours, even more preferably longer than 12 hours, in particular longer than 24 hours.

Accordingly, the present invention is also a method for diagnosing myocardial infarction, particularly non-ST elevation myocardial infarction, in a subject who exhibits signs and symptoms of acute coronary syndrome ACS but does not meet the diagnostic criteria for myocardial infarction. ,
a) measuring the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof in the subject sample;
b) comparing the amount of sFLT-1 or its variant measured in step a) with at least one reference amount, and c) based on the information obtained in step b), preferably obtained in steps a) and b) And diagnosing myocardial infarction, in particular non-ST elevation myocardial infarction, based on the obtained information.

The present invention further provides a method for diagnosing myocardial infarction, particularly non-ST elevation myocardial infarction, in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) comparing the amount of sFLT-1 or variant thereof measured in the sample of the subject with at least one reference amount, and b) based on the information obtained in step a) on myocardial infarction, in particular non-ST elevation myocardium It relates to a method comprising diagnosing an infarct.

The present invention further provides a method for diagnosing myocardial infarction, particularly non-ST elevation myocardial infarction, in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) to a method comprising diagnosing myocardial infarction, in particular non-ST elevation myocardial infarction, based on a comparison of the amount of sFLT-1 or variant thereof measured in the sample of the subject with at least one reference amount.

  Hepatocyte growth factor (HGF) was first identified and purified in 1984 and 1985 as an effective mitogen for primary cultured hepatocytes. HGF is a single-stranded precursor form, and further processing to a double-stranded form by a serine protease leads to its activation. Serine proteases involved in HGF activation include HGF activator or HGF converting enzyme and urokinase-type plasminogen activator (uPA). The receptor for HGF has been identified as the c-met proto-oncogene product. The c-Met receptor consists of a 50-kDa a-chain and a 145-kDa h-chain. When HGF binds to the c-Met receptor, tyrosine kinase activation is induced followed by phosphorylation of the C-terminal clustering tyrosine residue. HGF has an organtrophic role in the regeneration and protection of various organs including liver, lung, stomach, pancreas, heart, brain, and kidney. Hepatocyte growth factor regulates cell proliferation, cell motility, and morphogenesis by activating the tyrosine kinase signaling cascade after binding to the proto-oncogene c-Met receptor. Hepatocyte growth factor is secreted by mesenchymal cells and acts mainly on epithelial-derived cells as a multifunctional cytokine. Its ability to stimulate mitogenesis, cell motility, and matrix invasion gives it a central role in angiogenesis, tumorigenesis, and tissue regeneration. It is secreted as a single inactive polypeptide and is cleaved by a serine protease into a 69-kDa alpha chain and a 34-kDa beta chain. An active heterodimeric molecule is generated by a disulfide bond between the alpha and beta chains. This protein belongs to the S1 peptidase of the plasminogen subfamily, but has no detectable protease activity. Alternative splicing of this gene generates a number of transcriptional variants that encode different isoforms. The amino acid sequence of mouse HGF is shown in Genebank accession number NP034557.2, GI: 46048249. The amino acid sequence of human HGF is shown in Genebank accession number NP000592.3 GT33859835. Preferably, HGF represents human HGF. Furthermore, variants described in accordance with the present invention should have different amino acid sequences due to substitutions, deletions and / or additions of at least one amino acid, where the amino acid sequence of the variant is still the amino acid sequence of a particular HGF. It should be understood that preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% are identical. The variant may be an allelic variant, a splice variant, or any other species-specific homolog, paralog or ortholog. Furthermore, variants described herein include specific HGF or fragments of the above types of variants; however, these fragments have the essential immunological and biological properties described above. Preferably, the variant of HGF has immunological properties (ie, epitope composition) and / or biological properties comparable to those of human HGF. Thus, the variant should be recognized by the means or ligands used to measure cardiac troponin levels. Such a fragment may be, for example, a degradation product of HGF. In addition, different variants are included due to post-translational modifications such as glycosylation, phosphorylation or myristylation. Preferably, the biological property of HGF is the ability to bind to the proto-oncogene c-Met receptor.

  In yet another aspect of the present invention, measurements are made in an individual to diagnose myocardial infarction based on a comparison of the amount of hepatocyte growth factor (HGF) or variant thereof in a sample of the subject with at least one reference amount. The amount of HGF used is used. The method can include at least one of the following steps: a) measuring the amount of hepatocyte growth factor (HGF) or variant thereof in the subject sample, and b) HGF measured in step a). Or the amount of the variant is compared with at least one reference amount, and c) based on the information obtained in step b), preferably on the basis of the information obtained in steps a) and b). Diagnose ST-elevation myocardial infarction. This is particularly advantageous when the individual can be observed for a long time, preferably longer than 6 hours, more preferably longer than 8 hours, even more preferably longer than 12 hours, in particular longer than 24 hours.

Accordingly, the present invention is also a method for diagnosing myocardial infarction, particularly non-ST elevation myocardial infarction, in a subject who exhibits signs and symptoms of acute coronary syndrome ACS but does not meet the diagnostic criteria for myocardial infarction. ,
a) measuring the amount of soluble HGF or variant thereof in the subject sample;
b) comparing the amount of HGF or variant thereof measured in step a) with at least one reference amount, and c) based on the information obtained in step b), preferably information obtained in steps a) and b) To a method comprising the step of diagnosing myocardial infarction, in particular non-ST elevation myocardial infarction.

The present invention further provides a method for diagnosing myocardial infarction, particularly non-ST elevation myocardial infarction, in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) comparing the amount of HGF or variant thereof measured in the subject's sample with at least one reference amount, and b) diagnosing myocardial infarction based on the information obtained in step a).

The present invention further provides a method for diagnosing myocardial infarction, particularly non-ST elevation myocardial infarction, in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction,
a) to a method comprising diagnosing myocardial infarction, in particular non-ST elevation myocardial infarction, based on a comparison of the amount of HGF or variant thereof measured in the sample of the subject with at least one reference amount.

  The term “ANT-type peptide” includes pre-proANP, proANP, NT-proANP, and ANP variants thereof (see, for example, Bonow, 1996, Circulation 93: 1946-1950). Pre-propeptides include a short signal peptide that is cleaved enzymatically to release the propeptide. The propeptide is further cleaved into the N-terminal propeptide (NT-proANP) and the 28 amino acid active hormone ANP. ANP has a vasodilatory effect and drains water and sodium through the ureter.

  ANP has a shorter half-life than its inactive corresponding NT-proANP. ANP is produced exclusively in the atria and released from it. Thus, the amount of ANP may primarily reflect atrial function. The term “variant” in this regard relates to peptides that are substantially similar to ANP and NT-proANP. The term “substantially similar” is well understood by those skilled in the art. In particular, the variant may be an isoform or allele that exhibits amino acid exchange compared to the amino acid sequence of the most common peptide isoform in the human population. Furthermore, the variants described according to the invention should have a different amino acid sequence due to substitution, deletion and / or addition of at least one amino acid, wherein the amino acid sequence of the variant is still an amino acid of ANP or NT-proANP It should be understood that the sequence is preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical. The variant may be an allelic variant, a splice variant, or any other species-specific homolog, paralog or ortholog. Proteolytic products still recognized by diagnostic tools or ligands for each full-length peptide are also substantially similar; however, these fragments have the essential immunological and biological properties described above. Preferably, variants of ANP-type peptides have immunological properties (ie epitope composition) and biological properties comparable to those of the respective human ANP-type peptides. Thus, the variant should be recognized by the means or ligand used to measure the amount of ANP-type peptide. Such a fragment may be, for example, a degradation product of NT-proANP. In addition, different variants are included due to post-translational modifications such as glycosylation, phosphorylation or myristylation. Preferably, the biological properties of ANP are vasodilatory capacity and / or ability to excrete water and sodium.

  The term “variant” also refers to post-translationally modified peptides such as glycosylated peptides. A “variant” is also a peptide that has been modified after the collection of the sample, for example by covalently or non-covalently attaching a label, in particular a radioactive or fluorescent label, to the peptide. Measurement of the amount of peptide modified after collection of the sample is understood to be a measurement of the amount of original peptide that has not been modified.

In the context of the present invention, the ANP-type peptide is preferably NT-proANP.
The term “sample” refers to a sample of body fluid, a sample of separated cells, or a sample from a tissue or organ. Body fluid samples can be obtained by well-known techniques, preferably including blood, plasma, serum or urine samples, more preferably blood, plasma or serum samples. Tissue or organ samples can be obtained from any tissue or organ, for example, by biopsy. The separated cells can be obtained from a body fluid or tissue or organ by a separation method such as centrifugation or cell sorting. Preferably, the sample of cells, tissues or organs is obtained from cells, tissues or organs that express or produce the peptides described herein. Preferably, the term “sample” refers to a plasma or serum sample, more preferably a serum sample.

  In the present invention, the sample is taken at a suitable time known to those skilled in the art. Subjects to be tested should take into account that they are suffering from an acute pathophysiological condition (ACS) that requires prompt diagnosis and prompt decision on appropriate treatment. Preferably, the sample is from the subject according to the invention in a short time after the onset of acute coronary syndrome, for example after about 1 hour, not more than about 2 hours, not more than about 3 hours, not more than about 4 hours. , Not exceeding about 5 hours, or not exceeding about 6 hours. Preferably, the sample is taken immediately after the subject is presented to the physician (ie within a few minutes, ie within about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, or about 1 hour). It will be.

  In order to monitor the severity of ischemia, if the amount of sFLT1 and / or optionally HGF and / or optionally ANP-type peptide is measured repeatedly, preferably at least twice, 2 Each sample after and after will be taken at intervals that ensure effective monitoring of ischemic conditions. In general, the interval between each sample is about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 90 minutes, about 2 hours, about 3, 4, 5, or 6 hours. For example, the first sample is taken about 1 hour after the occurrence of signs and / or symptoms of acute coronary syndrome, or immediately after the subject is presented to the physician, and each subsequent sample is taken about 1 hour after the first sample. Collect. The number of samples taken will depend on the assessment of ischemia.

  Measurement of the amount of cardiac troponin, preferably troponin T, or sFLT-1, or HGF, or any of the other peptides or polypeptides or proteins described herein can be accomplished by measuring the amount or concentration. Preferably measuring quantitatively or semi-quantitatively. The terms polypeptide and protein are used interchangeably throughout the application. Measurements can be made directly or indirectly. Direct measurement involves measuring the amount of a peptide or polypeptide based on a signal obtained from the peptide or polypeptide itself, where the intensity of the signal is directly related to the number of molecules of that peptide or polypeptide in the sample. Correlate. Such a signal—sometimes referred to herein as an intensity signal—can be obtained, for example, by measuring the intensity value of a particular physical or chemical property of the peptide or polypeptide. For indirect measurements, measure the signal obtained from a secondary component (ie, a component that is not the peptide or polypeptide itself) or a biological readout system such as a measurable cellular response, ligand, label, or enzymatic reaction product For example.

According to the present invention, determination of the amount of peptide or polypeptide can be accomplished by any known means for measuring the amount of peptide in a sample. These means include immunoassay devices and methods, which can use the labeled molecules in a variety of sandwich, competition or other assay formats. Those assays will generate a signal indicative of the presence or absence of the peptide or polypeptide. Furthermore, the signal intensity can be correlated directly or indirectly (eg inversely proportional), preferably to the amount of polypeptide present in the sample. Still other suitable methods include measuring physical or chemical properties specific for the peptide or polypeptide, such as its exact molecular mass or NMR spectrum. Such methods preferably include biosensors, optical devices linked to immunoassays, biochips, analytical devices such as mass spectrometers, NMR analyzers, or chromatography devices. Further, the microplate ELISA-based methods in the way, fully-automated or robotic immunoassays (e.g., can be performed in Elecsys (TM) analyzers), CBA (an enzyme-type cobalt binding assay (C obalt B inding A ssay) , e.g. Roche-Hitachi (TM) analyzer), and latex agglutination assays (e.g., performed on the Roche-Hitachi (TM) analyzer).

  Preferably, the determination of the amount of peptide or polypeptide comprises the following steps: (a) cells capable of eliciting a cellular response whose intensity is indicative of the amount of the peptide or polypeptide are treated with the peptide or polypeptide for an appropriate period of time. Contact with the peptide and (b) measure its cellular response. To measure the cellular response, the sample or treated sample is preferably added to the cell culture and the internal or external cellular response is measured. A cellular response can include measurable reporter gene expression or secretion of a substance, such as a peptide, polypeptide or small molecule. This expression or substance should generate an intensity signal that correlates to the amount of peptide or polypeptide.

  Also preferably, measuring the amount of peptide or polypeptide comprises measuring a specific intensity signal obtained from the peptide or polypeptide in the sample. As noted above, such a signal is the signal intensity observed in the peptide or polypeptide specific m / z variable found in the mass spectrum or NMR spectrum specific for that peptide or polypeptide. There may be.

Measuring the amount of peptide or polypeptide preferably comprises the following steps: (a) contacting the peptide with a specific ligand, (b) (optionally) removing unbound ligand, (c ) Determine the amount of bound ligand. The bound ligand will generate an intensity signal. The bonds according to the present invention include both covalent and non-covalent bonds. A ligand according to the present invention may be any compound that binds to a peptide or polypeptide described herein, such as a peptide, polypeptide, nucleic acid, or small molecule. Preferred ligands include antibodies, nucleic acids, peptides or polypeptides, such as receptors or binding partners of said peptides or polypeptides, and fragments thereof containing the binding domains of those peptides, and aptamers, such as nucleic acid aptamers or peptide aptamers. It is. Methods for preparing such ligands are well known in the art. For example, identification and preparation of suitable antibodies or aptamers are also provided by the vendor. Those skilled in the art are aware of methods for developing derivatives of ligands with higher affinity or specificity. For example, random mutations can be introduced into nucleic acids, peptides or polypeptides. These derivatives can then be tested for binding according to screening methods known in the art, such as phage display. The antibodies described herein include both polyclonal and monoclonal antibodies, as well as fragments thereof that can bind an antigen or hapten, such as Fv, Fab and F (ab) ₂ fragments. The present invention also includes single chain antibodies and humanized hybrid antibodies in which the amino acid sequence of a non-human donor antibody exhibiting the desired antigen specificity is combined with the sequence of a human acceptor antibody. The donor sequence will usually include at least the antigen-binding amino acid residues of the donor, but may include other structural and / or functionally related amino acid residues of the donor antibody. Such hybrids can be prepared by several methods well known in the art. Preferably, the ligand or agent specifically binds to said peptide or polypeptide. Specific binding according to the present invention means that the ligand or agent should not substantially bind ("cross-react with") other peptides, polypeptides or substances present in the sample being analyzed. . Preferably, the specifically binding peptide or polypeptide binds with an affinity that is at least 3 times higher, preferably at least 10 times higher, even more preferably at least 50 times higher than any other related peptide or polypeptide. Should. Non-specific binding is acceptable if it can still be identified and reliably measured, for example by Western blotting according to its size or by its relatively high amount in the sample. Ligand binding can be measured by any method known in the art. Preferably the method is semi-quantitative or quantitative. A suitable method is described below.

First, ligand binding can be measured directly, eg, by NMR or surface plasmon resonance.
Second, if the ligand also acts as a substrate for the enzymatic activity of the peptide or polypeptide of interest, the enzyme reaction product can be measured (eg, the amount of protease determines the amount of cleaved substrate, eg, Western Can be measured by blotting). Alternatively, the ligand may exhibit enzymatic properties per se, and the “ligand / peptide or polypeptide” complex, ie the ligand to which each peptide or polypeptide is bound, is contacted with an appropriate substrate that can be detected by generation of an intensity signal. it can. For measurement of enzymatic reaction products, preferably the amount of substrate is saturated. Prior to the reaction, the substrate may be labeled with a detectable label. Preferably, the sample is contacted with the substrate for an appropriate period. An appropriate period represents the time required to produce a detectable, preferably measurable amount of product. Instead of measuring the amount of product, the time required for the appearance of a specified (eg detectable) amount of product can also be measured.

Third, the ligand can be covalently or non-covalently attached to a label that allows detection and measurement of the ligand. Labeling can be done by direct or indirect methods. The direct labeling method involves attaching the label directly (covalently or non-covalently) to the ligand. The indirect labeling method involves binding (covalently or non-covalently) a secondary ligand to a primary ligand. The secondary ligand should bind specifically to the primary ligand. The secondary ligand may be a target (receptor) of a tertiary ligand that binds to an appropriate label and / or binds to the secondary ligand. The use of secondary, tertiary, or higher order ligands is often employed to enhance the signal. Suitable secondary and higher order ligands can include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). The ligand or substrate can also be “tagged” with one or more tags known in the art. Such tags can then be targeted with higher order ligands. Suitable tags include biotin, digoxigenin, His-tag, glutathione-S-transferase, FLAG, GFP, myc-tag, influenza A virus hemagglutinin (HA), maltose binding protein, and the like. In the case of a peptide or polypeptide, the tag is preferably at the N-terminus and / or C-terminus. A suitable label is any label that can be detected by a suitable detection method. Typical labels include gold particles, latex beads, acridan esters, luminol, ruthenium, enzyme activity labels, radioactive labels, magnetic labels (eg “magnetic beads”, including paramagnetic and superparamagnetic labels) And fluorescent labels. Enzymatic activity labels include, for example, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, luciferase, and derivatives thereof. Substrates suitable for detection include di-amino-benzidine (DAB), 3,3′-5,5′-tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro- 3-indolyl-phosphate (4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate), available as ready-made stock from Roche Diagnostics), CDP-Star ™ (Amersham Biosciences), ECF (Trademark) (Amersham Biosciences). Appropriate enzyme-substrate combinations can produce colored reaction products, fluorescence or chemiluminescence, which can be measured according to methods known in the art (eg, photosensitive film or suitable camera). System use). The above-mentioned criteria are similarly applied to the measurement of enzyme reaction. Exemplary fluorescent labels include fluorescent proteins (eg, GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein, and Alexa dyes (eg, Alexa 568). Still other fluorescent labels are available from, for example, Molecular Probes (Oregon). The use of quantum dots as fluorescent labels is also considered. Exemplary radioactive labels include ³⁵ S, ¹²⁵ I, ³² P, ³³ P, and the like. The radioactive label can be detected by any known method, such as a light-sensitive film or a phosphor imager. Suitable measuring methods according to the present invention also include: precipitation methods (particularly immunoprecipitation methods), electrochemiluminescence (electrically generated chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme linked immunoassay). Solvent assay), sandwich enzyme immunoassay, electrochemiluminescence sandwich immunoassay (ECLIA), dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), scintillation proximity assay (SPA), turbidimetry ), Nephelometry, latex-enhanced turbidimetry or turbidimetry, or solid phase immunoassay. Still other methods known in the art (eg, gel electrophoresis, 2D gel electrophoresis, SDS polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and mass spectrometry), either alone or as described above Can be used in combination with other labeling methods or other detection methods.

  The amount of peptide or polypeptide can also preferably be determined according to the following: (a) contacting a solid support comprising a ligand for said peptide or polypeptide with a sample comprising the peptide or polypeptide; (B) Measure the amount of peptide or polypeptide bound to the support. Preferably the ligand selected from the group consisting of nucleic acids, peptides, polypeptides, antibodies and aptamers is preferably present in an immobilized form on a solid support. Materials for making solid supports are well known in the art, especially commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicon chips and surfaces, nitrocellulose strips Including membranes, sheets, duracyte, reaction tray wells and walls, plastic tubes, and the like. The ligand or agent can be bound to a wide variety of carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the present invention. Suitable methods for immobilizing / immobilizing the ligand are well known and include, but are not limited to, ionic, hydrophobic, covalent interactions, and the like. The use of “suspension arrays” is also considered as an array according to the invention (Nolan 2002, Trends Biotechnol. 20 (1): 9-12). In such suspension arrays, carriers, such as microbeads or microspheres, are present in suspension. The array consists of various microbeads or microspheres (which may be labeled) that carry various ligands. Methods for making such arrays, such as those based on solid phase chemistry and photolabile protecting groups, are generally known (US 5,744,305).

  Preferably, the blood obtained from the subject defined in the present invention is the amount of sFLT-1 and the amount of HGF (when measuring HGF) and the amount of ANP-type peptide, and optionally the amount of other peptides as measured in accordance with the present invention. It is measured in a sample, such as a serum or plasma sample. Preferably, such measurement is performed by ELISA. Such measurements by ELISA can be performed using, for example, the following: Quantikine human HGF immunoassay, R & D Systems, Inc. , Minneapolis, MN, and / or Quantikine human soluble VEGF Rl / Flt-1 immunoassay (for sFLT-1), R & D Systems, Inc. , Minneapolis, Minn., USA, and NT-proANP immunoassay, Biomedica Medizin product, Vienna, Austria. Optionally, the amount of each of sFLT-1 and / or HGF detailed elsewhere in this specification is the HBT ELISA test kit for human heart-type fatty acid binding protein (HyCult) for measuring the amount of H-FABP. Biotechnology, Uden, The Netherlands, and Tina-Quant® myoglobin test system (Roche Diagnostics) for measuring the amount of myoglobin, respectively.

  The term “amount” as used herein refers to an absolute amount (eg, sFLT-1 or HGF), a relative amount or concentration (eg, sFLT-1 or HGF), and any numerical value or Contains parameters. Such numerical values or parameters include intensity signal values derived from all specific physical or chemical properties obtained by direct measurement from said peptides, such as intensity values in mass spectra or NMR spectra. In addition, all numerical values or parameters obtained by indirect measurements detailed elsewhere herein, for example, expression levels measured from biological readout systems in response to said peptides, or specifically bound ligands The intensity signal obtained from is included. It should be understood that numerical values correlating to the above quantities or parameters can also be obtained by all standard arithmetic operations.

  As used herein, the term “compare” includes comparing the amount of peptide, polypeptide, protein contained in a sample to be analyzed to an appropriate reference source detailed elsewhere herein. . It should be understood that comparisons used herein represent comparisons of corresponding parameters or numerical values; for example, absolute amounts are compared to absolute reference amounts, while concentrations are compared to reference concentrations, or test samples Is compared with an intensity signal of the same type of the reference sample. The comparison described in step (b) of the method of the invention can be performed manually or with computer assistance. For computer-aided comparisons, measured values can be compared by computer program with values corresponding to appropriate criteria stored in a database. The computer program can further evaluate the results of the comparison, i.e. automatically provide the desired evaluation in an appropriate output format. Based on a comparison of the amount (s) measured in step a) and an appropriate reference amount (s), MI in the subject can be diagnosed. Comparing the amount of sFLT-1 measured in step (a) of the method of the invention with the reference amount for sFLT-1 detailed elsewhere in this application in step (b), and the amount of HGF in relation to HGF It should be understood to compare with a reference amount.

  Thus, as used herein, any of the terms “reference amount” is used in subjects with signs and symptoms of acute coronary syndrome and having a cardiac troponin amount that is less than an amount considered to be indicative of myocardial infarction (thus, Represents an amount by which an ischemic condition (which may also be referred to as “the degree of ischemia” in the context of the present invention) can be determined (in the subject matter of the invention). By comparison with a reference amount, it can be diagnosed whether the ischemia results in cardiac dysfunction / heart injury. The occurrence of the ACS symptoms has recently occurred. In this regard, recent onset means that the onset occurred preferably within 6 hours, more preferably within 4 hours, and most preferably within 2 hours, prior to taking a sample from the subject. Preferably, the baseline amount for ischemic severity is preferably within 6 hours, more preferably within 4 hours, and most preferably within 2 hours before taking a sample. A subject as defined in the present invention, diagnosed as having an onset of prognosis, ie, onset of MI, in particular NSTEMI, UAP, myocardial cessation, circulatory dysfunction, or CAD (no signs of ACS) It may be derived from a subject that has been made.

  In this aspect of the invention, the term “reference amount” as used herein refers to an ischemic condition in an individual, a physiologically healthy subject, or an ischemic condition that results in reversible myocardial infarction or irreversible heart injury. Represents the amount of polypeptide that can be diagnosed as being of a subject with.

  Thus, the reference amount will generally be derived from a subject known to be physiologically healthy or with an ischemic condition resulting in reversible myocardial infarction or irreversible heart injury.

  In all embodiments of the present invention, the amount of each marker used in the present invention (cardiac troponin, particularly troponin T; sFLT-1; HGF; and in some embodiments NT-proANP), which is an indicator of the ischemic state of the individual / Amount is measured by methods known to those skilled in the art.

In general, the amount (or amounts) of each peptide or peptides to determine each amount / amount or ratio (“threshold”, “reference amount”) that can establish the desired diagnosis according to each aspect of the present invention. / Quantity (s) or quantity ratio is determined in the appropriate patient group. Depending on the diagnosis to be established, the patient group can include, for example, only healthy individuals, or can include individuals suffering from a pathophysiological condition to be diagnosed as healthy individuals, or a condition to be diagnosed. Can include only individuals suffering from physiological conditions or include individuals suffering from various pathophysiological conditions to be distinguished by each marker (s) using validated analytical methods be able to. The results obtained are collected and analyzed by statistical methods known to those skilled in the art. The resulting threshold is then established according to the probability of suffering from the disease of interest and associated with a particular threshold. To establish the threshold value (s), reference value (s) or quantity ratio, for example, median of healthy and / or unhealthy patient population, 60 ^th , 70 ^th , 80 ^th , 90 ^th , 95 ^th , or even it may be useful to choose the 99 ^th percentile.

  A reference value for a diagnostic marker can be established and the amount of that marker in a patient sample can be easily compared to this reference value. The sensitivity and specificity of diagnostic and / or prognostic tests not only depend on the “quality” of the test analysis, but also on the definition of what constitutes an abnormal result. In fact, Receiver Operating Characteristic curves, or “ROC” curves, are generally calculated by plotting numerical values of variables against their relative frequencies in the “normal” and “disease” populations. The For any particular marker of the present invention, the distribution of marker amounts in subjects with and without disease may overlap. Under such conditions, the test cannot absolutely distinguish normal from the disease with a 100% accuracy rate, and the overlap area indicates the area where the test cannot distinguish normal from the disease. Above that (or below, depending on how the marker changes with the disease), the test is considered abnormal, below which the test is considered normal Select a threshold. The area under the ROC curve is a measure of the probability that the sensed measurement can properly identify the condition. The ROC curve can be used even when the test result does not necessarily give an accurate value. As long as the results can be ranked, ROC curves can be created. For example, test results for “disease” samples can be ranked according to degree (eg, 1 = low, 2 = normal, and 3 = high). This ranking can be correlated with results in the “normal” population to create an ROC curve. These methods are well known in the art. See, for example, Hanley et al, Radiology 143: 29-36 (1982).

  In certain embodiments, at least about 70% sensitivity, more preferably at least about 80% sensitivity, even more preferably at least about 85% sensitivity, even more preferably at least about 90% sensitivity, most preferably at least about 95. % Sensitivity, at least about 70% specificity, more preferably at least about 80% specificity, even more preferably at least about 85% specificity, even more preferably at least about 90% specificity, most preferably Select markers and / or marker panels that show at least about 95% specificity. In particularly preferred embodiments, both sensitivity and specificity are at least about 75%, more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, and most preferably at least about 95%. is there. The term “about” in this context represents ± 5% of the indicated measurement.

  In other embodiments, a positive likelihood ratio, a negative likelihood ratio, an odds ratio, or a hazard ratio, the test estimates the risk of the disease, or the disease Is used as a measure of the ability to diagnose. For positive likelihood ratios, a value of 1 indicates an equally positive outcome between subjects in both the “disease” and “control” groups; numbers greater than 1 are positive in the disease group A value of less than 1 indicates a more likely outcome; a value less than 1 indicates a greater likelihood of a positive outcome in the control group. For negative likelihood ratios, a value of 1 indicates an equally negative outcome between subjects in both the “disease” and “control” groups; numbers greater than 1 are negative in the test group A value of less than 1 indicates a more likely outcome; a value less than 1 indicates a more likely negative outcome in the control group. In certain preferred embodiments, the markers and / or marker panels are preferably at least about 1.5 or more, or about 0.67 or less, more preferably at least about 2 or more, or about 0.5 or less, even more preferably at least A positive likelihood ratio or negative of about 5 or more, or about 0.2 or less, more preferably at least about 10 or more, or about 0.1 or less, most preferably at least about 20 or more, or about 0.05 or less. It is selected to indicate the likelihood ratio. The term “about” in this context represents ± 5% of the indicated measurement.

  For odds ratio, a value of 1 indicates an equally positive outcome between subjects in both the “disease” group and the “control” group; a number greater than 1 indicates a positive outcome in the disease group A value less than 1 indicates a higher probability of a positive result in the control group. In certain preferred embodiments, the markers and / or marker panels are preferably at least about 2 or more, or about 0.5 or less, more preferably at least about 3 or more, or about 0.33 or less, even more preferably at least about 4 Or an odds ratio of about 0.25 or less, more preferably at least about 5 or more, or about 0.2 or less, most preferably at least about 10 or more, or about 0.1 or less. The term “about” in this context represents ± 5% of the indicated measurement.

  For risk ratios, a value of 1 indicates that the relative risk of endpoints (eg, death) is comparable between subjects in both the “disease” group and the “control” group; A value less than 1 indicates that the risk is greater in the control group. In certain preferred embodiments, the markers and / or marker panels are preferably at least about 1.1 or higher, or about 0.91 or lower, more preferably at least about 1.25 or higher, or about 0.8 or lower, even more preferably. Is a risk ratio of at least about 1.5 or more, or about 0.67 or less, more preferably at least about 2 or more, or about 0.5 or less, most preferably at least about 2.5 or more, or about 0.4 or less. Is selected. The term “about” in this context represents ± 5% of the indicated measurement.

  Exemplary panels are described herein, but one or more markers in these exemplary panels can be exchanged, added or deleted and still have clinically useful results. Panels are specific markers of disease (eg, markers that increase or decrease upon bacterial infection but do not increase or decrease in other disease states) and / or non-specific markers (eg, increased or decreased due to inflammation regardless of cause) Markers that decrease; markers that increase or decrease due to changes in blood flow blockage regardless of cause, etc.) can be included. While certain markers may not be critical in the methods described herein, the particular “fingerprint” pattern of change may actually serve as a specific indicator of disease state. As mentioned above, the pattern of change can be obtained from a single sample, or optionally taking into account temporary changes (or temporary changes in panel response values) in one or more members of the panel. Can be put.

In order to examine whether the patient suffering from the target disease is sufficiently reliably diagnosed by the selected reference value, the effectiveness (E) of the method of the present invention is determined for the reference value using, for example, the following equation: be able to:
E = (TP / TO) × 100;
Where TP = true positive, and TO = total number of tests = TP + FP + FN + TN, where FP = is false positive; FN = false negative, TN = true negative. E has a numerical value in the following range: 0 <E <100). Preferably, the value of E is at least about 50, more preferably at least about 60, more preferably at least about 70, more preferably at least about 80, more preferably at least about 90, more preferably at least about 95, more preferably at least If about 98, the reference value examined gives a sufficiently reliable diagnosis.

  Diagnosis of whether an individual is healthy or suffers from a particular pathophysiological condition can be made by established methods known to those skilled in the art. These methods vary depending on the pathophysiological state of the individual.

An algorithm for establishing a target diagnosis is shown in the application in the course of referring to each referenced aspect.
Accordingly, the present invention provides a method for determining a threshold amount indicative of a physiological state and / or a pathological state and / or a specific pathological state, wherein an appropriate marker (single or Also included is a method comprising measuring a plurality of quantities, collecting data and analyzing the data by statistical methods to establish a threshold.

The term “about” as used herein represents ± 20%, preferably ± 10%, preferably ± 5% of the indicated measured value or value.
The reference amount (threshold amount) relating to sFLT-1 which is an index of ischemic state defined in the present invention is as follows: about 92 pg / ml, more preferably about 109 pg / ml. An amount of sFLT-1 below the values shown above is indicative of a non-ischemic condition that is not associated with or resulting in reversible cardiac dysfunction or irreversible heart injury. An amount of sFLT-1 above the reference amount indicated above is indicative of an ischemic condition associated with or resulting in reversible cardiac dysfunction or irreversible heart injury.

  The reference amount (threshold amount) relating to HGF, which is an index of ischemia according to the present invention, is as follows: about 0.62 pg / ml, more preferably about 0.73 pg / ml. An amount of HGF below the number given above is indicative of a non-ischemic condition that is not associated with or resulting in reversible cardiac dysfunction or irreversible heart injury. An amount of HGF above the reference amount indicated above is indicative of an ischemic condition associated with or resulting in reversible cardiac dysfunction or irreversible heart injury.

  Preferably, if the measured sFLT-1, and optionally the HGF value, does not indicate an ischemic condition, the individual does not require any further testing for heart disease. In general, such individuals can return home.

  If an ischemic condition is diagnosed, each individual will need to be further examined for heart disease or cardiovascular disease. Accordingly, in a preferred embodiment, the present invention further comprises the step of further diagnosing the individual who has measured the amount of sFLT-1 and / or HGF. Still other suitable diagnostic methods in connection with the present invention include various types of stress tests, such as exercise ECG, exercise echocardiography, exercise computed tomography, exercise thallium scan; and angiography (invasive or virtual) For example, spiral computer tomography).

  Preferably, the reference amount (threshold amount) for NT-proANP for circulatory disorders as defined in the present invention is: about 1320 pg / ml, more preferably about 1673 pg / ml. An amount of NT-proANP that is greater than the numerical value shown above is an indicator of circulatory disturbance.

  It should be noted that figures for HGF and NT-proANP may change due to the development of an individual's chronic disease, such as heart failure, renal dysfunction or renal failure, and, in the case of HGF, acute or chronic liver disease It is. In the case of chronic disease, the values given above are significantly higher than the previously shown values that apply to individuals without chronic disease, especially not chronic heart disease, and also to acute coronary syndromes (eg, 25, (7, 8 or 10 times higher).

The term “at least one reference amount” means one or more than one reference amount, for example two reference amounts.
In a preferred embodiment of the present invention, the marker measurement described above is performed at intervals to determine the evaluation of the marker amount. This may be useful in assessing whether an acute event has occurred / has occurred.

  An acute event is presumed if at least one of the markers sFLT-1, HGF and / or NT-proANP is greater than a pre-determined reference amount. Preferably, the deviation is at least about 20%, or at least about 30%, or at least about 50%, or at least about 100%, more preferably at least about 200%, even more preferably at least about 500%, especially at least about 1000. %.

  The interval between two measurements of a particular marker (or markers) is at least about 15 minutes, at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 90 minutes, at least about 2 hours, at least about 3 hours. At least about 4 hours, at least about 5 hours, at least about 6 hours. Those skilled in the art are aware that this interval and deviation may vary according to the condition of the individual, the tendency of the amount of change for each marker (ie, the development of ischemia and / or circulatory disturbances), etc.

  Another aspect of the invention is to determine whether a subject suffers from myocardial infarction, in particular NSTEMI, after measuring cardiac troponin and / or measuring ECG, and step a) of the method of the invention measuring H-FABP and / or myoglobin prior to measuring the amount of sFLT-1 and optional HGF in b).

  An amount of myoglobin and optionally H-FABP that is greater than the reference amount to encompass the onset of MI in a subject defined in the present invention should be indicative of recent MI onset in that subject. An amount of myoglobin and optionally H-FABP that is less than the reference amount to rule out the onset of MI in a subject defined in the present invention has not recently developed MI myocardial infarction, and therefore the subject is affected by UAP. It should be an indicator that you may have In the context of the present invention, the amount of myoglobin that is the subject of the present invention is between the above reference amounts (a reference amount for including the recent onset of MI and a reference amount for excluding the recent onset of MI). It should be understood that some subjects need to be rediagnosed. Preferably, this measures the amount of both myoglobin and H-FABP, where both amounts do not match, eg one amount is greater (or less) than each reference amount, whereas the other It can also be done for subjects whose amount is not greater (or less) than each reference amount. In particular, an amount of myoglobin greater than 77 ng / ml in the subject defined in the present invention is an indicator of recent onset of MI (inclusion), whereas an amount less than 55 ng / ml is an indicator that MI has not recently occurred ( Excluded) Furthermore, the sensitivity and specificity of diagnosis based on the measurement of myoglobin in a sample of the subject defined in the present invention is at least related to H-FABP by measuring the amount of H-FABP in the sample of the subject in addition to the amount of myoglobin. When compared with one reference amount, it further increases. In particular, an amount of H-FABP greater than 5700 pg / ml is an indicator of recent onset of MI (inclusion) in subjects defined in the present invention, whereas an amount less than 2500 pg / ml is an indicator that MI has not recently occurred (Excluded).

  Myoglobin is a cytoplasmic heme protein consisting of a single-chain polypeptide of 154 amino acids and is almost exclusively expressed only in cardiomyocytes and oxidative skeletal muscle fibers. Like hemoglobin, myoglobin reversibly binds oxygen, thus promoting oxygen transport from erythrocytes to mitochondria during periods of increased metabolic activity, or acting as a reservoir for hypoxia or anoxia Ordway G. and Garry DJ, Myoglobin: an essential hemoprotein in striated muscle. 2004. Journal of Experimental Biology 207, 3441-3446 (2004).

  Heart-type fatty acid binding proteins (also referred to herein as H-FABP or cardiac fatty acid binding proteins) are small cytosolic proteins that move long-chain fatty acids in the myocardium from their cell membranes to their intracellular metabolic sites in the mitochondria ( Long-chain fatty acids then function as the primary transporter for transport to the citric acid cycle). It is generally believed that H-FABP is present in the myocardium and is rapidly released into the circulation in response to myocardial injury. Several studies have shown that H-FABP is an early biochemical marker of myocardial infarction; see, for example, Okamoto et al, Clin Chem Lab Med 38 (3): 231-8 (2000) Human heart-type cytoplasmic fatty acid-binding protein (H-FABP) for the diagnosis of acute myocardial infarction.Clinical evaluation of H-FABP in comparison with myoglobin and creatine kinase isoenzyme MB; O'Donoghue et al., Circulation, 114; 550-557 (2006) Prognostic Utility of Heart-Type Fatty Acid Binding Protein in patients with acute coronary syndrome; or Ruzgar et al, Heart Vessels, 21; 209-314 (2006) The use of human heart-type fatty acid-binding protein as an early diagnostic marker of myocardial necrosis in patients with acute coronary syndrome, and its comparison with troponin T and its creatine kinase-myocardial band).

  As used herein, myoglobin and H-FABP include variants of myoglobin and H-FABP polypeptides, respectively. Such variants have at least the same essential biological and immunological properties as specific myoglobin and H-FABP polypeptides. In particular, they can be detected by the same specific assays described herein, eg, ELISA assays using polyclonal or monoclonal antibodies that specifically recognize their myoglobin and H-FABP polypeptides, respectively. Share the same essential biological and immunological properties. Furthermore, variants described in accordance with the present invention should have different amino acid sequences due to substitutions, deletions and / or additions of at least one amino acid, wherein the amino acid sequence of the variant is still preferably a specific H-FABP. And at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of each of the and myoglobin polypeptides. It should be understood. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art. Preferably, the degree of identity is determined by comparing two sequences that are optimally aligned over the entire comparison window, wherein fragments of the amino acid sequences within the comparison window are compared to the reference sequence for optimal alignment. Can include additions or deletions (eg, gaps or overhangs) compared to (does not include additions or deletions). Determine the number of matching positions by determining the number of positions where the same amino acid residue appears in both sequences, divide the number of matching positions by the total number in the comparison window, and multiply the quotient by 100 to determine the sequence identity. Calculate the percentage by finding the percentage. The optimal alignment of the sequences for comparison was determined by the local homology algorithm of Smith and Waterman Add. APL. Math. 2: 482 (1981), Needleman and Wunsch J. Mol. Biol. 48: 443 ( 1970) homology alignment algorithm, Pearson and Lipman Proc. Natl. Acad Sci. (USA) 85: 2444 (1988) search for similarity method. Can be performed by computerized processing (GAP, BESTFIT, BLAST, PASTA, and TFASTA, in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.) Or by visual inspection. Once two sequences have been identified for comparison, GAP and BESTFIT are preferably used to determine their optimal alignment and thus the degree of identity. Preferably, default values of 5.00 for gap weight and 0.30 for gap weight length are used. The variants mentioned above may be allelic variants or any other species-specific homolog, paralog or ortholog. In addition, variants described herein include specific myoglobin and H-FABP polypeptides or fragments of the above types of variants; however, these fragments include the essential immunological and Has biological properties. Such fragments may be, for example, degradation products of myoglobin and H-FABP polypeptides. In addition, different variants are included due to post-translational modifications such as phosphorylation or myristylation. Preferably the biological property of myoglobin is its ability to reversibly bind oxygen. Preferably, the biological property of H-FABP is to transport long chain fatty acids from the cell membrane to their intracellular metabolic sites in the mitochondria, where the long chain fatty acids enter the citrate cycle.

  According to the method of the invention as described above and in the later stages, antibodies of sFLT-1, and preferably also of HGF, or means for measuring them are indicative of signs and symptoms of acute coronary syndrome and of myocardial infarction It should be understood that it can be used in the preparation of a diagnostic composition for diagnosing ischemia and the course of the severity of ischemia in a subject having an amount of cardiac troponin that is less than what is considered an indicator.

  The present invention also provides a method for stratifying or assessing the risk of suffering from myocardial infarction in a subject who exhibits signs and symptoms of acute coronary syndrome ACS but does not meet the diagnostic criteria for myocardial infarction, comprising: It relates to a method based on comparing the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof in a sample of interest with at least one reference amount. The method can include at least one of the following steps: a) measuring the amount of sFLT-1 or a variant thereof in the subject sample, b) sFLT-1 measured in step a) or the Comparing the amount of the variant with at least one reference amount, c) diagnosing ischemic conditions based on the information obtained in step b, preferably in steps a) and b), and d) in the information obtained in c) To assess the risk of suffering from myocardial infarction.

Accordingly, the present invention is also a method for stratifying or assessing the risk that a subject who exhibits signs and symptoms of acute coronary syndrome ACS but does not meet the diagnostic criteria for myocardial infarction will suffer from myocardial infarction, comprising:
a) measuring the amount of sFLT-1 or variant thereof in the subject sample;
b) comparing the amount of sFLT-1 or variant thereof measured in step a) with at least one reference amount;
c) diagnosing an ischemic condition based on information obtained in step b, preferably steps a) and b), and d) assessing the risk of suffering myocardial infarction based on the information obtained in c) Relates to a method comprising:

  The present invention also provides a method for identifying a subject (showing signs and symptoms of acute coronary syndrome ACS but not meeting diagnostic criteria for myocardial infarction) that may be subjected to cardiac intervention, the subject To a method based on comparing the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof in at least one reference amount. The method can include at least one of the following steps: a) measuring the amount of sFLT-1 or a variant thereof in the subject sample, b) sFLT-1 measured in step a) or the Comparing the amount of the variant with at least one reference amount, c) diagnosing ischemic conditions based on the information obtained in step b, preferably in steps a) and b), and d) in the information obtained in c) To assess the risk of suffering from myocardial infarction.

Thus, the present invention further provides a method for identifying a subject who may receive a cardiac intervention, wherein the subject exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction. There,
a) measuring the amount of sFLT-1 or variant thereof in the subject sample;
b) comparing the amount of sFLT-1 or variant thereof measured in step a) with at least one reference amount;
c) a method comprising diagnosing an ischemic condition based on information obtained in step b), preferably steps a) and b), and d) identifying a subject based on the information obtained in c) .

  Furthermore, the present invention also recommends or determines a possible cardiac intervention or a possible cardiac intervention in a subject who exhibits signs and symptoms of acute coronary syndrome ACS but does not meet the diagnostic criteria for myocardial infarction To a method based on comparing the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof in a sample of the subject to at least one reference amount. The method can include at least one of the following steps: a) measuring the amount of sFLT-1 or a variant thereof in the subject sample, b) sFLT-1 measured in step a) or the Compare the amount of the variant with at least one reference amount, c) recommend or decide to start a cardiac intervention based on the information obtained in c), start the cardiac intervention or avoid the cardiac intervention.

Therefore, the present invention recommends or determines a possible cardiac intervention or initiates a potential cardiac intervention in a subject who exhibits signs and symptoms of acute coronary syndrome but does not meet the diagnostic criteria for myocardial infarction A method for
a) measuring the amount of sFLT-1 or variant thereof in the subject sample;
b) comparing the amount of sFLT-1 or variant thereof measured in step a) with at least one reference amount;
c) Diagnosing ischemic conditions based on the information obtained in step b), preferably steps a) and b), and d) recommending or deciding to start cardiac intervention based on the information obtained in c) Relates to a method comprising initiating or avoiding a cardiac intervention.

All the aspects shown above, a method for stratifying or assessing the risk that a subject will suffer from myocardial infarction, a method for identifying subjects who are likely to undergo cardiac intervention, and potential in the subject A method for recommending or determining a cardiac intervention or initiating a potential cardiac intervention (subject presents with signs and symptoms of acute coronary syndrome but does not meet diagnostic criteria for myocardial infarction) The method can also be carried out using the following steps:
a) diagnosing an ischemic condition based on measuring the amount of sFLT-1 or variant thereof in the subject's sample and comparing the amount of sFLT-1 or variant thereof to at least one reference amount; And b) based on the information obtained in step a)
bi) assessing the risk that the subject will suffer from myocardial infarction; and / or bii) identifying a subject who may receive cardiac intervention; and / or biii) recommending or determining potential cardiac intervention in the subject Or initiate a possible cardiac intervention.

Furthermore, the present invention provides all the aspects set forth above, a method for stratifying or assessing the risk that a subject will suffer from myocardial infarction, a method for identifying a subject that may undergo cardiac intervention, and A method for recommending or deciding a possible cardiac intervention in a subject or initiating a potential cardiac intervention (subject presents signs and symptoms of acute coronary syndrome but meets myocardial infarction diagnostic criteria A) using the following steps for each method:
ai) assessing the subject's risk of suffering from myocardial infarction; and / or aii) identifying subjects who may receive cardiac intervention; and / or aiii) recommending or determining potential cardiac intervention in the subject Or initiate a possible cardiac intervention;
In that case, all stages ai), aii) and / or aiii) measure the amount of sFLT-1 or variant thereof in the sample of the subject and determine the amount of sFLT-1 or variant thereof at least one reference Based on diagnosing the ischemic state of a subject by comparing with the amount.

  In the subject of the method of the invention as described above, the additional step aa) further measures the amount of hepatocyte growth factor (HGF) or variant thereof in the subject's sample, and in step bb) at least one reference amount for HGF; Compare. Thus, in step c), a measured amount of sFLT-1 or a variant thereof and HGF or a variant thereof, and a comparison between the amount of sFLT-1 and at least one reference amount for sFLT-1, and an amount of HGF and at least for HGF An ischemic condition is diagnosed based on a comparison with one reference amount. Preferably, the amount of sFLT-1 and then the amount of HGF are measured first, but the amount of sFLT-1 and HGF is determined in any order, ie simultaneously or first sFLT-1, then HGF, or first HGF, then sFLT It is also contemplated to measure -1.

  In yet another aspect of the present invention, all the aspects set forth above, ie, a method for stratifying or assessing the risk of a subject suffering from myocardial infarction, to identify a subject that may receive cardiac intervention And a method for recommending or determining a possible cardiac intervention in a subject or initiating a potential cardiac intervention (subject presents signs and symptoms of acute coronary syndrome but diagnoses myocardial infarction Measure the amount of hepatocyte growth factor (HGF) or variant thereof instead of sFLT-1. In the subject of this method of the invention, in an additional step, the amount of sFLT-1 or variant thereof in the subject's sample is further measured and at least one reference amount for sFLT-1 to diagnose ischemic conditions Compare with

  In a preferred embodiment, the amount of cardiac troponin is measured simultaneously with the measurement of sFLT-1 and / or HGF; the measurement of the amount of cardiac troponin precedes the measurement of sFLT-1 and / or HGF Also good. Thus, the ischemic state of an individual is preferably determined by measuring the amount of sFLT-1 and / or HGF, or optionally still other markers, simultaneously with or after the measurement of troponin. Thus, if the amount of cardiac troponin is measured prior to the measurement of sFLT-1 and / or HGF, the measurement of the amount of sFLT-1 and / or HGF is postponed until the amount of cardiac troponin is known, and myocardial infarction The amount of sFLT-1 and / or HGF is measured only when the amount of troponin is less than the amount generally recognized in the art as an indicator of MI, particularly NSTEMI. The amount of cardiac troponin can even be zero, i.e. not detectable with currently available tests.

  In yet another preferred embodiment, the ECG of each subject is measured simultaneously with the sFLT-1 measurement and / or the HGF measurement; the ECG measurement may be performed prior to the sFLT-1 measurement and / or the HGF measurement. . Accordingly, the ischemic state of the individual is determined by measuring the amount of sFLT-1 and optionally HGF, preferably simultaneously with or after the ECG measurement. Thus, if the ECG measurement is performed prior to the measurement of sFLT-1 and / or HGF, the measurement of the amount of sFLT-1 and / or HGF may be postponed until the ECG is recorded. Preferably, the amount of sFLT-1 and / or HGF is measured only when the subject's ECG does not show ST elevation. Preferably, if the ECG shows ST elevation, the subject will be considered as suffering from STEMI; this is generally performed according to the method of the invention—for the purpose of diagnosing ischemic conditions prior to MI— It means not.

  Said preferred embodiment of the method of the invention can be implemented to measure only the level of cardiac troponin or to measure only ECG; alternatively, the method of the invention comprises measuring the level of cardiac troponin Both ECG measurements are included.

The reference amounts of sFLT-1 and HGF are shown in advance to serve as an index of the ischemic state of the individual.
The reference amount (threshold amount) relating to sFLT-1 which is an index of ischemic condition defined in the present invention is as follows: about 92 pg / ml, more preferably about 109 pg / ml. An amount of sFLT-1 less than that indicated above is indicative of a non-ischemic condition that is not associated with or resulting in reversible cardiac dysfunction or irreversible heart injury. An amount of sFLT-1 or a variant thereof above the reference amount indicated above is indicative of an ischemic condition associated with or resulting in reversible cardiac dysfunction or irreversible heart injury.

  The reference amount (threshold amount) relating to HGF for the ischemic state defined in the present invention is as follows: about 0.62 pg / ml, more preferably about 0.73 pg / ml. An amount of HGF less than that indicated above is indicative of a non-ischemic condition that is not associated with or resulting in reversible cardiac dysfunction or irreversible heart injury. An amount of HGF or a variant thereof above the reference amount indicated above is indicative of an ischemic condition associated with or resulting in reversible cardiac dysfunction or irreversible heart injury.

  If sFLT-1, and optionally HGF values, do not indicate an ischemic condition, the individual does not require any further testing for heart disease. In general, such individuals can return home.

  When diagnosed with an ischemic condition by the amount of sFLT-1 and, optionally, HGF, in a preferred embodiment of the invention, each individual receives a treatment regimen also referred to as “cardiac intervention” as detailed below.

  If an ischemic condition is diagnosed by the amount of sFLT-1, and optionally HGF, each individual will need to be further examined for cardiovascular or cardiovascular disease. In a preferred embodiment, these further diagnostic methods in connection with the present invention include various types of stress tests such as exercise load, eg exercise echocardiography, exercise computed tomography, exercise thallium scan; and angiography ( Invasive or virtual, such as spiral computed tomography. Depending on the results of this further examination, each individual will receive a treatment plan, also referred to as “cardiac intervention”, detailed below.

In yet another preferred embodiment of the invention, the amount of NT-proANP is measured in order to diagnose a potential circulatory disorder in the subject.
The reference amount for ANP-type peptides, particularly NT-proANP, was previously indicated in relation to the reference amount (threshold amount) for NT-proANP for the circulatory disturbance defined in the present invention, and is about 1320 pg. / Ml, more preferably about 1674 pg / ml. An amount of NT-proANP that is greater than the numerical value shown above is an indicator of circulatory disturbance.

  Another aspect of the invention is to determine whether the subject is already suffering from myocardial infarction, in particular NSTEMI, after cardiac troponin measurement and / or ECG measurement, and step a) of the method of the invention measuring H-FABP and / or myoglobin prior to measuring the level of sFLT-1 and optional HGF in b).

  An amount of myoglobin and optionally H-FABP that is greater than the reference amount to encompass the onset of MI in a subject defined in the present invention should be indicative of recent MI onset in that subject. An amount of myoglobin and optionally H-FABP that is less than the reference amount to rule out the onset of MI in a subject defined in the present invention has not recently developed MI myocardial infarction, and therefore the subject is affected by UAP. It should be an indicator that you may have In the context of the present invention, the amount of myoglobin that is the subject of the present invention is between the above reference amounts (a reference amount for including the recent onset of MI and a reference amount for excluding the recent onset of MI). It should be understood that some subjects need to be rediagnosed. Preferably, this measures the amount of both myoglobin and H-FABP, where both amounts do not match, eg one amount is greater (or less) than each reference amount, whereas the other It can also be done for subjects whose amount is not greater (or less) than each reference amount.

  Thanks to the method, risk / success stratification can be easily performed before cardiac intervention is performed on the patient. If it turns out that a patient is unlikely to receive a cardiac intervention, that dangerous, time-consuming and / or expensive therapy can be avoided. Thus, the method of the present invention would be beneficial to the health system in that it saves resources in addition to preventing subjects from the harmful and serious side effects associated with cardiac intervention.

  With respect to the method of the invention described above, a subject who is not suffering from MI but is associated with or diagnosed to develop an ischemic condition that is expected to result in reversible cardiac dysfunction or irreversible cardiac injury. It should be understood that there is a possibility of undergoing cardiac intervention.

  As used herein, the term “identify” means assessing whether a subject is likely to receive cardiac intervention. As will be appreciated by those skilled in the art, such an assessment is usually not intended to be accurate for all (ie 100%) subjects to be identified. The term, however, requires that a statistically significant portion of subjects can be identified (eg, a cohort in a cohort study). Whether a part is statistically significant is further determined by those skilled in the art using various well-known statistical evaluation tools such as confidence interval determination, p-value determination, Student's t-test, Mann-Whitney test, etc. It can be judged without doing. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. More preferably, at least 60%, at least 70%, at least 80%, or at least 90% of a group of subjects can be properly identified by the methods of the invention.

  The term “cardiac intervention” includes treatment regimes deemed appropriate by those skilled in the art. The term includes surgical procedures that affect the cardiovascular system and preferably the heart, microsurgical procedures, interventions by other invasive therapies, and conservative (non-surgical) therapeutic methods. Conservative methods are known in the art and include non-pharmacological and pharmacological methods. A pharmacological method relates to the administration of a medicament. Suitable medicaments include: ACE inhibitors, especially enalapril, captopril, ramipril, trandolapril; angiotensin receptor antagonists and aldosterone antagonists, especially losartan ( Losartan, Valsartan, Irbesartan, Candesartan, Telmisartan, Eprosartan, Spironolactone; statins, especially atorvastatin, fluvastatin, fluvastatin Lovastatin, pravastatin, rosuvastatin, simvastatin; beta-blockers such as proprenolol, metoprolol (meto) prolol), bisoprolol, carvedilol, bucindolol, nebivolol; nitrates; adrenergic agonists such as dobutamine, dopamine, epinephrine, isoprotenolol (Isoprotenerol), norepinephrine, phenylephrine; antiplatelet drugs, especially aspirin and clopidrogel; anticoagulants, especially warfarin, heparin, thrombin inhibitors, thrombolytic drugs. Preferably, the cardiac intervention as used herein is a treatment plan aimed at restoring the proper oxygen supply of the heart. This is preferably accomplished by restoring blood flow in the blood vessels supporting the heart, i.e. the entire coronary blood vessel. These blood vessels can be damaged, for example, due to thrombotic or atheromatous plaques. Thus, cardiac intervention should preferably include the destruction and / or removal of such plaques and, if necessary, vascular repair. Preferred cardiac interventions according to the present invention include percutaneous coronary angioplasty, percutaneous transluminal coronary balloon angioplasty aimed at restoring blood flow, patency of blood vessels, plaque stabilization, and / or reducing intracoronary thrombus load. Selected from the group consisting of surgery, laser angioplasty, coronary stent implantation, bypass grafting or intraluminal procedures.

  When an individual is suffering from circulatory disturbances (indicated by elevated levels of ANP-type natriuretic peptides), the circulatory disorders generally resolve when the cause underlying ischemia is treated.

Yet another aspect relates to the diagnosis of myocardial infarction in a subject. This method for diagnosing myocardial infarction in a subject comprises at least one of the following steps:
a) measuring the amount of natriuretic peptide and / or troponin T in the subject's sample;
b) comparing the amount measured in step a) with a reference amount; and c) preferably based on the information obtained in step d), preferably based on the information obtained in steps a) and b) Diagnose.

  Cardiac troponin (troponin T or variant thereof, and troponin I or variant thereof), NT-proANP or variant thereof, and to a lesser extent sFLT-1 or variant thereof, HGF or variant thereof, H-FABP or variant thereof Variants and the concentrations indicated in this application for myoglobin or variants thereof are those for patients suffering from renal dysfunction, preferably those suffering from renal failure, especially those suffering from chronic and end-stage renal failure. Those skilled in the art know that is not applicable. In a preferred embodiment of the invention, patients suffering from renal dysfunction, preferably patients suffering from renal failure, in particular patients suffering from chronic and end-stage renal failure are not included in the method of the invention. (Excluded). In other preferred embodiments, patients with renal hypertension are not included (excluded) in the methods of the invention. Preferably, a “subject” as used herein is a patient suffering from renal dysfunction, preferably a patient suffering from renal failure, particularly a patient suffering from chronic and end-stage renal failure, more preferably Excludes patients with renal hypertension, most preferably all patients suffering from any of the diseases or conditions described in this document. In this regard, “renal failure” is considered to have a glomerular filtration rate (GFR) below the normal range of 60-120 ml / min, preferably below 60 ml / min. Chronic renal failure is a persistent progressive decline in renal function, often resulting in end-stage renal failure. End stage renal failure is diagnosed when the GFR reaches a rate of about 30 ml / min. GFR is measured by creatinine clearance, which is known to those skilled in the art. Subjects with impaired renal function are associated with higher levels of troponin I and troponin T than those indicated above; this is due to impaired clearance of this peptide. This level varies with the severity of kidney damage.

The severity of kidney damage can be divided into various grades as shown below:
0: ≧ 90 ml / min 1: ≧ 90 ml / min; with proteinuria 2: ≧ 60˜ <90 ml / min 3: ≧ 30˜ <60 ml / min 4: ≧ 15˜ <30 ml / min 5: <15 ml / Min
(Source: National Kidney Foundation, Am J. Kidney Dis 39 suppl 1, 2002; published in Clinical Practice Guidelines for chronic kidney disease).

  The present invention further includes means for determining the amount of sFLT-1 and optionally HGF in a sample of interest, and means for comparing the amount to at least one reference amount. A kit or device for performing the method is included.

  The term “kit” as used herein refers to a collection of said means, which are preferably provided individually or in a single container. The kit can further include a means for measuring the amount of cardiac troponin. Optionally, the kit can further comprise a user manual for interpreting the results of any measurement (s) with respect to the diagnosis of the ischemic condition of the subject as defined in the present invention. In particular, such a manual can contain information about which measured quantity corresponds to which type of diagnosis. This is detailed elsewhere in this specification. In addition, such user manuals can provide instructions on the proper use of kit components for the measurement of the amount of each biomarker.

  As used herein, the term “device” includes a set of means, including at least the means linked together, so that an ischemic condition can be diagnosed or an object that may be subjected to cardiac intervention can be identified. About. The device of the present invention may further comprise means for measuring the amount of cardiac troponin. Preferred means for measuring the amount of sFLT-1 and HGF, as well as means for performing the comparison, are disclosed above in connection with the method of the present invention. The way in which these means are operatively linked will depend on the type of means included in the device. For example, when applying means for automatically measuring the amount of peptide, the data obtained by these automatically operating means can be processed, for example, by a computer program to obtain the desired result. Preferably, in such a case, these means are constituted by a single device. The device thus comprises an analytical unit for measuring the amount of peptide or polypeptide in the applied sample and a computer unit for processing the obtained data for evaluation. Alternatively, if a means such as a test strip is used to measure the amount of peptide or polypeptide, the means for comparison can include a control strip or table that assigns the measured amount to a reference amount. The test strip is preferably bound to a ligand that specifically binds to the peptide or polypeptide described herein. The strip or device preferably includes a means for detecting binding of the peptide or polypeptide to its ligand. Preferred means for detection are disclosed in connection with the above aspects relating to the method of the present invention. In such cases, the means are operatively linked in that the user of the system links the measurement result of that quantity with its diagnostic or prognostic value by instructions and interpretation given in the manual. . In such embodiments, these means can be provided as separate devices and are preferably packaged together as a kit. One skilled in the art will know how to connect these means without difficulty. Preferred devices are those that can be applied without special knowledge of a specialist, such as test strips or electronic devices that only need to be loaded with a sample. The result may be obtained as raw data output that the physician needs to interpret. However, the output of the device is the raw data processed, ie, evaluated, and does not require a physician to interpret it. Still other preferred devices are the analysis units / devices described above according to the method of the invention (eg, biosensors, arrays, solid supports, plasmon surface resonance devices coupled to ligands that specifically recognize sFLT-1 or HGF) , NMR spectrometer, mass spectrometer, etc.) or evaluation unit / device.

  The invention also provides a kit or device for measuring the amount of sFLT-1 and optionally HGF in a sample of interest, and / or a means for measuring the amount of sFLT-1 and optionally HGF. And / or a means for comparing the amount of sFLT-1 and optionally the amount of HGF with at least one reference amount for the following purposes: Although showing signs and symptoms of acute coronary syndrome Diagnosing ischemic condition in a subject who does not meet diagnostic criteria for myocardial infarction; and / or monitoring the severity of ischemic condition in the subject; and / or stratifying the risk that the subject will suffer from myocardial infarction Identify subjects that may be subject to cardiac intervention; and / or potential subjects in that subject That decide to heart intervention.

  The invention also provides at least one antibody against sFLT-1 and optionally HGF, and / or means for measuring the amount of sFLT-1 and optionally HGF, and / or sFLT-1 and optionally A means for selectively comparing the amount of HGF with at least one reference amount relates to the preparation of a diagnostic composition for: Myocardial infarction showing signs and symptoms of acute coronary syndrome Diagnosing an ischemic condition in a subject that does not meet the diagnostic criteria of; and / or monitoring the severity of the ischemic condition in the subject; and / or stratifying the risk of the subject suffering from myocardial infarction / Assess; and / or identify a subject that may receive cardiac intervention; and / or may be in that subject Decide to organs intervention.

All references cited herein are incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned herein.
The following examples merely illustrate the invention. They should in no way be construed as limiting the scope of the invention.

In the following examples, the following test methods were used to determine the amount of each peptide:
Troponin T was measured using an ELECSYS 2010 analyzer with a highly sensitive hs troponin T immunoassay test method by Roche Diagnostics. First incubation: 50 μm sample, biotinylated monoclonal anticardiac troponin T specific antibody, and monoclonal anticardiac troponin T specific antibody labeled with a ruthenium complex (Ru (bpy) ²⁺ ₃ ) react to form a sandwich complex Form the body. Second incubation: After the addition of streptavidin-coated microparticles, the complex becomes bound to the solid phase by the interaction of biotin and streptavidin. The reaction mixture is aspirated into the measuring cell, where the microparticles are captured on the surface of the electrode by magnetism. Unbound material is then removed. Generation of chemiluminescence is induced by applying a voltage to the electrode, and measurement is performed with a photomultiplier tube. The result is judged by a calibration curve.

  HGF (hepatocyte growth factor) was tested by enzyme-linked immunoassay (RD Systems, Minneapolis, catalog Nr.HG00) using monoclonal antibodies specific for HGF and precoated microplates. Standards and samples are pipetted into the wells and any HGF present is bound by the immobilized antibody. After washing away unbound material, an enzyme-linked polyclonal antibody specific for HGF is added to the wells. After washing away unbound antibody-enzyme reagent, a substrate solution is added to the well, and color develops in proportion to the amount of HGF bound in the first step. Stop color development and measure color intensity.

  NT-proANP was measured with the proANP ELISA assay (B1-20892) determined from Biomedica, Austria, Vienna. This sandwich assay includes a polyclonal sheep NT-proANP specific antibody bound to a microtiter strip. The sample is added to the microtiter strip so that proANP can bind to the antibody. After proANP has bound to this first antibody, a second proANP specific antibody is added to the container. This second antibody is conjugated with horseradish peroxidase (HRP). After incubation, unbound enzyme-conjugated antibody is removed by washing the microtiter strip. Finally, tetramethylbenzidine (TMB) is added as a substrate for HRP. The higher the sample contains proANP, the more conjugated antibody will bind. Thus, the total activity of HRP present in the container depends on the amount of proANP in the sample, and the initial TMB conversion is a measure of the amount of NT-proANP in the sample.

  sFlt-1 was measured with the sFlt-1 immunoassay used with the Roche Diagnostics, Elecsys and COB AS analyzers from Mannheim, Germany. This assay is based on the sandwich principle and includes two monoclonal sFlt-1 specific antibodies. Of these, the first is biotinylated and the second is labeled with a tris (2,2'-hypyridyl) ruthenium (TT) complex. In the first incubation step, both antibodies are incubated with the sample. A sandwich complex comprising sFlt-1 and two different antibodies is formed. In the next incubation step, streptavidin-coated beads are added to the complex. The beads bind to the sandwich complex. The reaction mixture is then aspirated into the measuring cell, where the beads are captured magnetically on the surface of the electrode. Next, voltage is applied to the electrode to induce the generation of chemiluminescence from the ruthenium complex, and this is measured with a photomultiplier tube. The amount of light depends on the amount of sandwich complex on the electrode.

The test methods listed above are preferably employed for the present invention in general for the measurement of each peptide.
Example 1. Measurement of baseline values 149 people (mean age 42,8 years) were tested for the presence of sFlT1, proANP and HGF; they were asymptomatic at the time of the test and within 2 weeks prior to it, in particular they also had chest pain There was also no evidence of cardiac dysfunction shown by the NT-proBNP test.

  Test results (see Table 1):

In the ^{context of the} present invention, preferably the 95th percentile is considered a suitable reference value: individuals exhibiting marker values above these are considered suffering from an ischemic condition. More preferably, it defines a reference value 99 ^th percentile is indicative of ischemia. Respect NT-proANP, preferably ^{95 th} percentile, more preferably ^{99 th} percentile is considered as a reference value that defines a numerical value indicative of similarly circulatory disorders.

Example 2: sFlT-1, HGF and NT-proANP in a patient with chest pain and suspected acute coronary syndrome
A total of 33 patients (mean age 62, 47-72), 21 men and 12 women showing signs and symptoms of acute coronary syndrome within 2 hours after onset in the emergency room were included in the study. 14/33 had a family history of coronary artery disease, 21/33 had established coronary artery disease, and 5/33 reported previous myocardial infarction. All patients had normal renal function as measured by serum creatinine, and H-FABP and myoglobin were below cut-off values for future myocardial infarction (MI) (see: WO 2008/145689, WO 2009/033831 ). Patients included in the analysis were those who did not meet the NON-STEMI criteria at the end of follow-up. Troponin levels were below 99 percent (0.14 ng / ml, at the time of arrival and at the recommended time interval (at least 6-9 hours after arrival) in all samples measured. The mean of 0.079 ng / ml) is that all patients have the Joint ESC / ACCF / AHA / WHF Task Force for the Redefinition of Myocardial Infarction (The Joint European Society of Cardiology / American College of Cardiology Committee: Universal definition of myocardial infarction, European Heart Journal (2007), cited above).

  All patients showed no increase in myoglobin, H-FABP or NT-proBNP (± 20%) during the course of the observation period. Therefore, none of the patients selected for this study showed myocardial infarction according to current guidelines. A change in the level of sFlT1 / HGF was seen in 27 patients and no change occurred in 6 patients, because 27 patients had biochemical evidence of ischemia and in 6 patients Indicates that there was no such indication. An acute change in circulatory function, indicated by changes in NT-proANP concentration, was observed in 11 patients. Acute changes in circulatory function were seen only in patients with laboratory evidence of ischemia.

Serum samples were collected by venipuncture, centrifuged within 30 minutes, and serum supernatants were stored at −20 ° C. until analysis.
The test was performed according to the manufacturer's instructions.

Table 2 shows the changes in sFlT1 and / or HGF seen in 33 patients with acute coronary syndrome and not developing myocardial infarction.
Table 3 represents the individual results of the selected patients.

  Patient No. 14 presented biochemical evidence of ischemia as shown by increased concentrations of HGF and sFlT1. During the course of the observation, the levels of sFlT1 and HGF decreased, which is an indication that biochemical evidence of ischemia has been reversed, consistent with pain remission without further medication in this patient (patient Nitroglycerin spray was given by an emergency physician before arriving in the emergency room). There was a slight increase in proANP levels during the observation period, indicating that there were no significant acute functional changes caused by this event.

  Patient No. 21 presented with signs and symptoms of ACS, which recovered for about 5 minutes when visiting the emergency room, sFlT1 increased slightly on arrival, then decreased and increased again, which was observed during the observation period Consistent with chest pain. Early chest pain was accompanied by significant functional changes, as indicated by a significant increase in proANP levels immediately upon arrival. This was consistent with ventricular tachycardia (about 30 seconds) seen in the ambulance when transported to the emergency room.

  Patient No. 40 presented with a severe ischemic episode as confirmed by elevated and increasing sFlT1 and HGF levels compared to the baseline dose set forth above, and this patient was one hour in the emergency room It also showed evidence of acute functional changes as indicated by a significant increase in later proANP levels. With nitroglycerin spray, the symptoms of ischemia disappeared, biochemical evidence of ischemia disappeared, and the patient did not develop myocardial infarction.

  Patient No. 42 was admitted with signs and symptoms of ACS and had sFlT1 and HGF levels that were significantly elevated at the time of arrival compared to the baseline dose set forth above. During follow-up, sFlT1 and HGF gradually decreased, indicating that there was moderate ischemia following initial severe ischemia and eventually resolved. There was a slight increase in proANP at the time of arrival and at 24 hours during follow-up.

  Patient No. 43 presented with signs and symptoms of ACS, which were no longer present upon arrival in the emergency room. He was accompanied by slightly elevated sFlT1 and HGF levels compared to the baseline dose set forth above, but there were no definite biochemical signs of ischemia. His proANP level did not change throughout the observation period. The patient was known to have coronary artery disease, but based on clinical symptoms he was ultimately deemed to have noncardiac symptoms.

  Patient No. 52 had an ACS that still existed when it arrived in the emergency room. Treating him with a nitroglycerin spray cleared his symptoms. During ECG monitoring in the emergency room, no significant arrhythmia was observed. The biochemical evidence of ischemia was reversible, as evidenced by sFlT1 and HGF measurements (marker levels increased or decreased with the extent of ischemia). There was a slight change in proANP levels while arriving in the emergency room.

  These patients did not develop myocardial infarction.

Comparative Example 1: sFlT-1, HGF and NT-proANP in patients with MI
The 22 patients who developed myocardial infarction were the Joint ESC / ACCF / AHA / WHF Task Force for the Redefinition of Myocardial Infarction (The Joint European Society of Cardiology / American College of Cardiology Committee: Universal definition of myocardial infarction, European Heart 4 patients (including patient 2, Table 4) had troponin T concentrations above the 99% percent of the normal reference population, which were associated with troponin T concentrations that did not meet the criteria of Journal (2007), cited above) Was accompanied. This study simply explains that the reference population developing MI has increased sFlT1 and HGF compared to the healthy reference population outlined in Table 1 for all patients developing MI. (This is used as a “positive control”).

  In all patients, sFlT1, HGF and proANP are increased compared to the reference dose defined above at the time of study entry, and two such patients are shown in Table 4. Six patients were diagnosed with STEMI (ie, at the end of follow-up, the Joint ESC / ACCF / AHA / WHF Task Force for the Redefinition of Myocardial Infarction (The Joint European Society of Cardiology / American College of Cardiology Committee: Universal definition of myocardial infarction, European Heart Journal (2007), cited above). All other patients were accompanied by NON-STEMI according to the Joint ESC / ACCF / AHA / WHF Task Force for the Redefinition of Myocardial Infarction.

Patient No. 1 : Presenting with signs and symptoms of ACS, he was accompanied by elevated sFlT1 and HGF levels compared to the reference dose defined above at the time of arrival, which declined during follow-up, followed by signs of ischemia Disappeared. At 4 hours after arrival at the hospital, troponin T levels increased and NT-proANP as an indication of acute functional changes. Since troponin T levels were elevated, coronary angiography was performed and the patient was implanted with a stent.

Patient No. 2 : Presented with signs and symptoms of ACS, which were still present upon arrival in the emergency room. On ECG he had ST elevations in II, III and aVF, indicating myocardial infarction in the posterior wall of the left ventricle. He had sFlT1 and HGF levels that were significantly elevated compared to the reference amount defined above, and troponin T was increased compared to the reference amount defined above, but at this point it was diagnosed as non-STEMI. There wasn't. He was scheduled to immediately angiographically and a second sample was taken immediately before the intervention, which increased NT-proANP and troponin T and continuously increased HGF and sFlT1 compared to the reference amount defined above. Showed the level. A stent was implanted during angiography to re-establish coronary flow.

  The results are shown in Table 4 below.

  These data show that more severe ischemia results in higher amounts of sFlT1 and HGF (STEMI vs. NSTEMI). sFLT-1 and HGF give short-term information about ischemic conditions, as can be seen from the fact that their respective amounts decrease even after several hours in STEMI patients. These data further indicate that ischemia can cause circulatory dysfunction as indicated by elevated NT-proANP levels.

Claims (13)

Method for detecting an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but whose troponin T level in the subject sample is less than 0.1 ng / ml and does not meet the diagnostic criteria for myocardial infarction Because
a) measuring the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof in the subject sample;
b) comparing the amount of sFLT-1 or its variant measured in step a) with at least one reference amount, and c) detecting an ischemic condition based on the information obtained in step b).   2. The method of claim 1, further comprising measuring the amount of hepatocyte growth factor (HGF) or a variant thereof and comparing to a reference value for HGF.   The reference amount of sFLT-1 or a variant thereof serving as an index of ischemia is about 92 pg / ml, and the amount of sFLT-1 or a variant thereof below the reference amount is a reversible cardiac dysfunction or irreversible heart. An amount of sFLT-1 or a variant thereof that is an indicator of non-ischemic conditions that is not associated with or does not cause injury, and that is greater than a reference amount is associated with reversible cardiac dysfunction or irreversible heart injury, or The method according to claim 1, wherein the method is an indicator of ischemic condition.   The reference amount of HGF or a variant thereof is about 0.62 pg / ml, wherein a sub-reference amount of HGF or a variant thereof is not associated with or causes reversible cardiac dysfunction or irreversible heart injury. A non-ischemic condition indicator, an amount of HGF or a variant thereof greater than a reference amount is an indicator of ischemic condition associated with or resulting in reversible cardiac dysfunction or irreversible heart injury, The method according to claim 1. The method further comprises to determine the circulatory disorder in a subject, comprising measuring the amount of ANP-type peptide in a sample The method according to any one of claims 1 to 4. The method according to claim 5, wherein the ANP-type peptide is NT-proANP or a variant thereof.   7. The method of claim 6, wherein an amount of NT-proANP or variant thereof greater than about 1320 pg / ml is an indicator of circulatory disturbance.   8. The method of any one of claims 1-7, wherein the method further comprises measuring each subject's ECG.   The method according to any one of claims 1 to 8, wherein the method further comprises measuring myoglobin or a variant thereof and / or cardiac fatty acid binding protein (HFABP) or a variant thereof in the sample. Method for monitoring an ischemic condition in a subject who exhibits signs and symptoms of acute coronary syndrome but whose troponin T level in the subject's sample is less than 0.1 ng / ml and does not meet the diagnostic criteria for myocardial infarction Because
a) measuring the amount of soluble fms-like tyrosine kinase-1 (sFLT-1) or variant thereof in the subject sample at at least two different time points, and b) sFLT-1 or variant thereof measured in step a) And c) detecting ischemic conditions at at least two different time points based on the information obtained in step b) and thereby monitoring the ischemic condition A method comprising stages. A method for identifying a subject that may receive cardiac intervention, wherein the subject exhibits signs and symptoms of acute coronary syndrome, but the amount of troponin T in the subject's sample is 0.1 ng / Less than ml does not meet the diagnostic criteria for myocardial infarction,
a) measuring the amount of sFLT-1 or variant thereof in the sample of the subject; and b) comparing the amount of sFLT-1 or variant thereof measured in step a) with at least one reference amount;
c) detecting an ischemic condition based on the information obtained in step b), and d) identifying the object based on the information obtained in step b). Recommendations for possible cardiac intervention in subjects who show signs and symptoms of acute coronary syndrome but do not meet the diagnostic criteria for myocardial infarction with a troponin T level of less than 0.1 ng / ml in the subject sample A method of providing information on which to base a decision or possible cardiac intervention,
a) measuring the amount of sFLT-1 or variant thereof in the sample of the subject; and b) comparing the amount of sFLT-1 or variant thereof measured in step a) with at least one reference amount;
c) detecting an ischemic condition based on the information obtained in step b), and d) recommending or determining a possible cardiac intervention or possible heart based on the information obtained in step b) Provide information on which the intervention begins,
A method involving that.   Administration of medications with cardiac interventions aimed at restoring blood flow, patency of blood vessels, plaque stabilization, and / or reducing intracoronary thrombus load; percutaneous coronary angioplasty; percutaneous transluminal coronary balloon angioplasty 13. The method of claim 12, selected from the group of surgery; laser angioplasty; coronary stent implantation; bypass grafting or intraluminal procedures.